Nucleic acid amplification primers for detecting cytokeratins and examination method with the use of the primers

ABSTRACT

It is intended to provide novel primers to be used in gene amplification reactions for detecting human cytokeratins (Human CK). A primer containing an oligonucleotide comprising a base sequence which is selected from among the the 270- to 1375-th regions of the base sequence represented by SEQ ID NO:1 and the regions of complementary strands thereof and represented by, for example, any of SEQ ID NOS:2 to 341 is constructed. Further, a primer containing an oligonucleotide comprising a base sequence which is selected from among the 270- to 930-th regions of the base sequence represented by SEQ ID NO:342 and the regions of complementary strands thereof and represented by, for example, any of SEQ ID NOS:343 to 434 is constructed. Furthermore, a primer containing an oligonucleotide comprising a base sequence which is selected from among the 340 to 490-th regions or the 495- to 1050-th regions of the base sequence represented by SEQ ID NO:435 and the regions of complementary strands thereof and represented by, for example, any of SEQ ID NOS:436 to 474 is constructed.

TECHNICAL FIELD

The present invention relates to nucleic acid amplifying primers fordetection of human cytokeratins.

BACKGROUND ART

Cytokeratins (hereinafter, referred to as “CK”) are proteins formingfibrous backbone of a cell and form a family of at least 20 genes. Assuch cytokeratins, human CK18, human CK19, human CK20 and the like areknown. CKs are expressed in epithelial cells.

For example, previous reports described that human CK18 is expressed notonly in normal tissues such as mammary gland, lung, large intestine,stomach but also in cancerous tissues, while human CK18 is not expressedin lymph nodes which are not epithelial tissues. As to human CK19,previous reports described that it is expressed in lung cancer, stomachcancer, breast cancer, pancreatic cancer, prostate cancer and the like,and difference in expression level of human CK19 is observed betweennormal tissues and cancerous tissues. As to human CK20, previous reportsdescribed that it is expressed in colon cancer, stomach cancer, Merkelcell cancer, gynaecologic mucus cancer, transitional cell cancer,pancreatic cancer and bile duct cancer, and also in this case,difference in expression level of human CK20 is observed between normaltissues and cancerous tissues.

In consideration of the above facts, it is possible to knowpresence/absence of metastasis of cancer by examination ofpresence/absence of expression of human CK18, human CK19 and/or humanCK20 in tissues such as lymph nodes. Also it is possible to knowpresence/absence of metastasis of cancer by searching for those exhibitdifference in expression level between normal tissues and canceroustissues.

A cancer cell leaving the primary lesion site will metastasize aroundthe body through the blood stream and lymphatic system. In a surgery ofcancer, accurate detection of metastasis and proper treatment based onthe extent of metastasis are required so as to remove lesions asreliable as possible. For this reason, intraoperative diagnosis ofcancer metastasis to lymph nodes has great significance. For example, inthe case of breast cancer, the range to be removed is getting smallerfor improving the QOL, and diagnosis for lymph node metastasis duringthe surgery can be significant guidelines for determining the minimum oflymph node dissection. In the case of esophageal cancer, detection of asite where lymph node metastasis occurs may give guidelines forselection and determination of operative procedures includingventrotomy, thoracotomy and collar incision. In the case of prostatecancer, presence of lymph node metastasis will give guidelines fordecision to conduct hormonal therapy while stopping the prostatectomy.Also in the case of stomach cancer, it will give guidelines forselecting an operative procedure and therapeutic strategy after thesurgery. Considering the burden on a patient, intraoperative diagnosisfor cancer metastasis should be conducted rapidly.

One procedure for diagnosing cancer metastasis to lymph nodes isdetecting CK proteins which are tumor markers. For example, this isachieved by freezing a resected lymph node and staining a section of thefrozen tissue. However, this procedure is accompanied with a risk thatmicrometastasis is overlooked because only the information about thesection is relied on.

Recent development in gene analysis technique has enabled effectivecancer diagnoses through detection of expression of tumor marker genes.For example, PCR technique enables a target DNA fragment to be amplifiedto several hundreds times by repetition of dissociation of DNA strandsto a single-stranded DNA, binding of primers sandwiching a specificregion in the DNA strand, and synthesis of DNA by DNA polymerase (seeJP-A 61-274697), and can be used as a high-sensitive analyzing techniquefor nucleic acids in various types of sample. For example, since the PCRtechnique can analyze nucleic acids in samples obtained from animal bodyfluids or tissues, it is useful for diagnosis of infection diseases,genetic diseases, cancers and the like.

For detection of RNA, RT-PCR technique can be used. the RT-PCR techniqueinvolves extracting RNA, for example, from tumor tissues; synthesizingcDNA by the help of reverse transcriptase (RT) using oligo(dT) or randomhexamer as a primer; amplifying the cDNA using PCR technique fordetection. The exemplary case of diagnosis of fibroblast tumor using theRT-PCR technique has been reported (Hokkaido Igaku Zasshi p. 135-141,Vol. 66(2), (1991)). The RT-PCR makes it possible to detect expressionof mRNA of CK from ablated tissues, so that the problem that cancermetastasis is overlooked can be avoided to some extent. In the field ofdiagnosis of tumor or cancer, such nucleic acid amplifying methods havebeen put into practical use (“Kanai's manual of clinical laboratorymedicine” the 31st ed., pp. 1314, KANEHARA & Co., LTD., published onSep. 20, 1998).

However, the PCR technique necessitates an operation for denaturing atemplate DNA from double-strand DNA to single-strand, as well asrequires repetitive amplifying reactions under plural temperatureconditions. Furthermore, in general, it takes about two hours to obtaina detectable amplified product, so that it was not desirable as anintraoperative test that requires rapidity.

As a DNA amplifying method other than the PCR technique, LAMP method hasbeen reported (refer to International Publication WO00/28082). The LAMPmethod is a gene amplifying method using a plurality of primersincluding a primer that forms a hairpin structure at a terminal of theamplified product as the strand displacement reaction proceeds. In aninitial reaction, using two kinds of inner primers (FIP, RIP) and twokinds of outer primers (F3 primer, R3 primer) and a strand displacingDNA polymerase, a dumbbell-like structure having a single-strand loop ateach end is synthesized from a template DNA. Starting from thisstructure, the amplification cycle carried out so that extension andsynthesis of DNA proceed on the DNA itself as a temperate, from the 3′end of this structure. The amplified product comprises a repeatedstructure of plural units, and each unit comprises a set ofcomplementary regions in the same strand that forms a region to beamplified sandwiched between the primers wherein two nucleic acids haveinverted base sequences. The LAMP method does not require the operationfor denaturing a template DNA from double strand to single strand byheating, and is characterized by continuous process of amplificationunder a constant temperature (refer to Bio Venture, Vol. 1, p. 109-115(2001) and BIO INDUSTRY, Vol. 18, No. 2, p. 15-29 (2001)). When thetemplate is RNA, it is possible to synthesize the starting structure ina similar manner by adding a reverse transcriptase to the composition ofthe reaction mixture, whereby amplification can be proceeded (RT-LAMPmethod). According to the LAMP method, a sufficient amount of amplifiedproduct for detection can be obtained in about 30 minutes. Therefore,the time required for detection is reduced, so that it can be appliedfor diagnosing cancer metastasis to lymph nodes for the purpose of rapiddetermination of the therapeutic strategy, for example. Furthermore,since a result can be acquired rapidly, application to theintraoperative diagnosis is also expected.

The basic concept of primers applied to the LAMP method can be found inInternational Publication No. WO00/28082 and International PublicationNO. WO02/24902.

Primers and probes to be used in PCR for detection of human CK18 havebeen already reported (refer to Gene, 159(1), p. 43-47(1995)). Likewise,primers or probes to be used in PCR for detection of human CK19 havealso been reported (refer to U.S. Pat. No. 6,203,992 and Breast CancerResearch and Treatment 60, p. 143-151(2000)) Also primers or probes tobe used in PCR for detecting human CK20 have been reported (refer toBritish J. of Cancer, 77(8), p. 1327-1332(1998) and British J. ofCancer, 82(1), p157-160 (2000)).

However, nobody have reported primers to be applied to LAMP methodintended for detection of human CKs, and there is a need to develop suchprimers. In addition, as for the primers to be applied to other nucleicacid amplifying means, there is a need to construct a new primer or aprimer set that is useful for detection in addition to known primers.

(Document Of Conventional Art)

Patent document 1: International Publication No. WO00/28082

Patent document 2: International Publication No. WO02/24902

Patent document 3: U.S. Pat. No. 6,203,992

Non patent document 1: Bio Venture, Vol. 1, p. 109-115 (2001)

Non patent document 2: BIO INDUSTRY, Vol. 18, No. 2, p. 15-29 (2001)

Non patent document 3: Gene, 159(1), p. 43-47(1995)

Non patent document 4: Breast Cancer Research and Treatment 60, p.143-151(2000)

Non patent document 5: British J. of Cancer, 77(8), p. 1327-1332(1998)

Non patent document 6: British J. of Cancer, 82(1), p157-160 (2000)

DISCLOSURE OF THE INVENTION

(Problems to be Solved by the Invention)

It is an object of the present invention to provide new primers for usein a nucleic acid amplifying reaction for detecting human CKs. Morespecifically, the present invention is directed to provide primers fornucleic acid amplification according to the LAMP method.

(Means for Solving the Problems)

The inventors have diligently researched for overcoming the aboveproblems, and finally constructed primers for nucleic acid amplificationwhich can be effectively applied to the LAMP method and used fordetecting human CKs.

That is, the present invention includes:

1. A primer for nucleic acid amplification for detecting cytokeratinbased on the LAMP method;

2. The primer for nucleic acid amplification according to item 1,wherein the cytokeratin is selected from the group consisting ofcytokeratin 18, cytokeratin 19 and cytokeratin 20;

3. A primer for nucleic acid amplification for detecting humancytokeratin 18 comprising an oligonucleotide having a sequence selectedfrom the following groups 1) to 5);

1) an oligonucleotide selected from a region of base position 270-1375in a base sequence of SEQ ID NO: 1 and a complementary strand regionthereof, containing at least 5 or more successive bases in SEQ ID NO.1and/or in the complementary strand thereof,

2) an oligonucleotide having a base sequence represented by any one ofSEQ ID NOs: 2 to 341,

3) a complementary strand of the oligonucleotide according to theabove 1) or 2),

4) an oligonucleotide capable of hybridizing with the oligonucleotideaccording to any one of the above 1) to 3) under stringent conditions,and

5) an oligonucleotide comprising a base sequence obtainable from theoligonucleotides according to the above 1) to 4) wherein one to severalbases are replaced, inserted, added or otherwise mutated, theoligonucleotide having a primer function;

4. A primer for nucleic acid amplification for detecting humancytokeratin 18, comprising an oligonucleotide selected from the basesequences of SEQ ID NOs: 66 to 88 or 179 to 341;

5. The primer for nucleic acid amplification according to the above 3.or 4., wherein nucleic acid amplification is conducted by the LAMPmethod;

6. A primer set for nucleic acid amplification for detecting humancytokeratin 18, wherein at least two kinds of primers are selected fromprimers for nucleic acid amplification, each primer comprising anoligonucleotide comprising a sequence selected from the followinggroups 1) to 4);

1) an oligonucleotide selected from a region of base position 270-1375in a base sequence of SEQ ID NO: 1 and a complementary strand regionthereof, containing at least 5 or more successive bases in SEQ ID NO.1and/or in the complementary strand thereof,

2) an oligonucleotide having a base sequence represented by any one ofSEQ ID NOs: 2 to 341,

3) a complementary strand of the oligonucleotide according to theabove 1) or 2),

4) an oligonucleotide capable of hybridizing with the oligonucleotideaccording to any one of the above 1) to 3) under stringent conditions,and

5) an oligonucleotide comprising a base sequence obtainable from theoligonucleotides according to the above 1) to 4) wherein one to severalbases are replaced, inserted, added or otherwise mutated, theoligonucleotide having a primer function;

7. The primer set for nucleic acid amplification for detecting humancytokeratin 18 according to the above 6., wherein the nucleic acidamplification is conducted by the LAMP method;

8. The primer set for nucleic acid amplification for detecting humancytokeratin 18 according to the above 7., wherein at least four kinds ofprimers are selected from the primers for nucleic acid amplificationeach comprising an oligonucleotide;

9. The primer set for nucleic acid amplification for detecting humancytokeratin 18 according to any one of the above 6. to 8., wherein atleast two kinds of primers included in the primer set respectivelyrecognize two regions of the base sequence of SEQ ID NO: 1 and/or acomplementary strand thereof;

10. The primer set according to any one of the above 7. to 9., whereinthe primers included in said primer set recognize at least 6 regions inthe base sequence of SEQ ID NO:1 and/or the complementary strandthereof;

11. A primer set comprising a combination of primers, one of the primersbeing selected from (a) SEQ ID NOs: 234 to 286, the other selected from(b) SEQ ID NOs: 287 to 341, among the primers for nucleic acidamplification for detecting human cytokeratin 18, each comprising anoligonucleotide selected from the base sequences of SEQ ID NOs:234 to341;

12. The primer set according to the above 11., further comprising acombination of primers, one of the primers being selected from (c) SEQID NOs: 66 to 88, the other selected from (d) SEQ ID NOs: 179 to 201,among the primers for nucleic acid amplification for detecting humancytokeratin 18 each comprising an oligonucleotide selected from the basesequences of SEQ ID NOs: 66 to 88 or SEQ ID NOs: 179 to 201;

13. The primer set according to the above 11. or 12., further comprisinga combination of primers, one of the primers being selected from (e) SEQID NOs: 202 to 219, the other selected from (f) SEQ ID NOs: 220 to 233,among the primers for nucleic acid amplification for detecting humancytokeratin 18 each comprising an oligonucleotide selected from the basesequences of SEQ ID NOs: 202 to 233;

14. The primer set for nucleic acid amplification for detectingcytokeratin 18 comprising either one of the following groups 1) to 4);

1) a primer set including oligonucleotides having base sequences of SEQID NOs: 234, 287, 66 and 179 as primers,

2) a primer set including oligonucleotides having base sequences of SEQID NOs: 252, 297, 68 and 182 as primers,

3) a primer set including oligonucleotides having base sequences of SEQID NOs: 259, 307, 72 and 184 as primers,

4) a primer set including oligonucleotides having base sequences of SEQID NOs: 278, 331, 79 and 193 as primers;

15. The primer set for nucleic acid amplification for detecting humancytokeratin 18 comprising either one of the following groups 1) to 4);

1) a primer set including oligonucleotides having base sequences of SEQID NOs: 234, 287, 66, 179, 203 and 220 as primers,

2) a primer set including oligonucleotides having base sequences of SEQID NOs: 252, 297, 68, 182, 211 and 223 as primers, 3) a primer setincluding oligonucleotides having base sequences of SEQ ID NOs: 259,307, 72, 184, 212 and 226 as primers,

4) a primer set including oligonucleotides having base sequences of SEQID NOs: 278, 331, 79, 193, 214 and 228 as primers;

16. The primer set for nucleic acid amplification for detecting humancytokeratin 18 comprising either one of the following groups 1) to 8);

1) a primer set including oligonucleotides having base sequences of SEQID NOs: 280, 334, 82 and 195 as primers,

2) a primer set including oligonucleotides having base sequences of SEQID NOs: 281, 335, 83 and 196 as primers,

3) a primer set including oligonucleotides having base sequences of SEQID NOs: 282, 336, 84 and 197 as primers,

4) a primer set including oligonucleotides having base sequences of SEQID NOs: 282, 337, 84 and 197 as primers,

5) a primer set including oligonucleotides having base sequences of SEQID NOs: 283, 338, 85 and 198 as primers,

6) a primer set including oligonucleotides having base sequences of SEQID NOs: 284, 339, 86 and 199 as primers,

7) a primer set including oligonucleotides having base sequences of SEQID NOs: 285, 340, 87 and 200 as primers,

8) a primer set including oligonucleotides having base sequences of SEQID NOs: 286, 341, 88 and 201 as primers;

17. The primer set for nucleic acid amplification for detecting humancytokeratin 18 comprising either one of the following groups 1) to 6);

1) a primer set including oligonucleotides having base sequences of SEQID NOs: 282, 336, 84, 197, 216 and 229 as primers,

2) a primer set including oligonucleotides having base sequences of SEQID NOs: 282, 337, 84, 197, 216 and 230 as primers,

3) a primer set including oligonucleotides having base sequences of SEQID NOs: 282, 337, 84, 197, 216 and 231 as primers,

4) a primer set including oligonucleotides having base sequences of SEQID NOs: 283, 338, 85, 198, 217 and 232 as primers,

5) a primer set including oligonucleotides having base sequences of SEQID NOs: 286, 341, 88, 201, 218 and 233 as primers,

6) a primer set including oligonucleotides having base sequences of SEQID NOs: 286, 341, 88, 201, 219 and 233 as primers;

18. A method for detecting nucleic acids of human cytokeratin 18 using anecessary primer selected from the primers according to the above 3. or4., or a primer set selected from the primer sets according to any oneof the above 5. to 17.;

19. A primer for nucleic acid amplification for detecting humancytokeratin 19 comprising an oligonucleotide having a sequence selectedfrom the following groups 1) to 5);

1) an oligonucleotide selected from a region of base position 270-930 ina base sequence of SEQ ID NO: 342 and a complementary strand regionthereof, containing at least 5 or more successive bases in SEQ ID NO.1and/or in the complementary strand thereof,

2) an oligonucleotide having a base sequence represented by any one ofSEQ ID NOs: 343 to 432,

3) a complementary strand of the oligonucleotide according to theabove 1) or 2),

4) an oligonucleotide capable of hybridizing with the oligonucleotideaccording to any one of the above 1) to 3) under stringent conditions,and

5) an oligonucleotide comprising a base sequence obtainable from theoligonucleotides according to the above 1) to 4) wherein one to severalbases are replaced, inserted, added or otherwise mutated, theoligonucleotide having a primer function;

20. A primer for nucleic acid amplification for detecting humancytokeratin 19, comprising an oligonucleotide selected from the basesequences of SEQ ID NOs: 357 to 361 or 378 to 434;

21. The primer for nucleic acid amplification for detecting humancytokeratin 19 according to the above 19. or 20., wherein the nucleicacid amplification is conducted by the LAMP method;

22. A primer set for nucleic acid amplification for detecting humancytokeratin 19, wherein at least two kinds of primers are selected fromprimers for nucleic acid amplification, each primer comprising anoligonucleotide comprising a sequence selected from the followinggroups 1) to 5);

1) an oligonucleotide selected from a region of base position 270-930 ina base sequence of SEQ ID NO: 342 and a complementary strand regionthereof, containing at least 5 or more successive bases in SEQ ID NO.342and/or in the complementary strand thereof,

2) an oligonucleotide having a base sequence represented by SEQ ID NOs:343 to 434,

3) a complementary strand of the oligonucleotide according to theabove 1) or 2),

4) an oligonucleotide capable of hybridizing with the oligonucleotideaccording to any one of the above 1) to 3) under stringent conditions,and

5) an oligonucleotide comprising a base sequence obtainable from theoligonucleotides according to the above 1) to 4) wherein one to severalbases are replaced, inserted, added or otherwise mutated, theoligonucleotide having a primer function;

23. The primer set for detecting human cytokeratin 19 according to theabove 22., wherein the nucleic acid amplification is conducted by theLAMP method;

24. The primer set for nucleic acid amplification for detecting humancytokeratin 19 according to the above 22. or 23., wherein at least fourkinds of primers are selected from the primers for nucleic acidamplification each comprising an oligonucleotide;

25. The primer set for nucleic acid amplification for detecting humancytokeratin 19 according to any one of the above 22. to 24., wherein atleast two kinds of primers included in the primer set respectivelyrecognize two regions of the base sequence of SEQ ID NO: 342 and/or acomplementary strand thereof;

26. The primer set for detecting human cytokeratin 19 according to anyone of the above 22. to 25., wherein the primers included in said primerset recognize at least six regions in the base sequence represented bySEQ ID NO:342 and/or a complementary strand thereof;

27. A primer set comprising a combination of primers, one of the primersbeing selected from (a) SEQ ID NOs: 413 to 417, 419, 422 or 424 to 427,the other selected from (b) SEQ ID NOs: 418, 420, 421, 423 or 428 to434, among the primers for nucleic acid amplification for detectinghuman cytokeratin 19, each comprising an oligonucleotide selected fromthe base sequences of SEQ ID NOs: 413 to 434;

28. The primer set according to the above 27., further comprising acombination of primers, one of the primers being selected from (c) SEQID NOs: 357 to 361, the other selected from (d) SEQ ID NOs: 378 to 384,among the primers for nucleic acid amplification for detecting humancytokeratin 19 each comprising an oligonucleotide selected from the basesequences of SEQ ID NOs: 357 to 361 or SEQ ID NOs: 378 to 384;

29. The primer set according to the above 27. or 28., further comprisinga combination of primers, one of the primers being selected from (e) SEQID NOs: 385 to 398, the other selected from (f) SEQ ID NOs: 399 to 412,among the primers for nucleic acid amplification for detecting humancytokeratin 19 each comprising an oligonucleotide selected from the basesequences of SEQ ID NOs: 385 to 412;

30. A primer set comprising either one of the following combinations 1)to 4);

1) combination of primers selected respectively one from (a) SEQ ID NO:413 to 417, (b) SEQ ID NO: 418, (c) SEQ ID NO: 357 and (d) SEQ ID NO:378,

2) combination of primers selected respectively one from (a) SEQ ID NO:419, (b) SEQ ID NO: 420 to 421, (c) SEQ ID NO: 358 and (d) SEQ ID NO:379,

3) combination of primers selected respectively one from (a) SEQ ID NO:422, (b) SEQ ID NO: 423, (c) SEQ ID NO: 359 and (d) SEQ ID NO: 380,

4) combination of primers selected respectively one from (a) SEQ ID NO:424 to 427, (b) SEQ ID NO: 428 to 434, (c) SEQ ID NO: 360 to 361 and (d)SEQ ID NO: 381 to 384, among primers for nucleic acid amplification fordetecting human cytokeratin 19 each comprising an oligonucleotide havinga base sequence represented by each SEQ ID NO;

31. A primer set comprising either one of the following combinations 1)to 4);

1) combination of primers selected respectively one from (a) SEQ ID NO:413 to 417, (b) SEQ ID NO: 418, (c) SEQ ID NO: 357, (d) SEQ ID NO: 378,(e) SEQ ID NO: 385 to 391 and (f) SEQ ID NO: 399 to 402,

2) combination of primers selected respectively one from (a) SEQ ID NO:419, (b) SEQ ID NO: 420 to 421, (c) SEQ ID NO: 358, (d) SEQ ID NO: 379,(e) SEQ ID NO: 392 to 393 and (f) SEQ ID NO: 403 to 406,

3) combination of primers selected respectively one from (a) SEQ ID NO:422, (b) SEQ ID NO: 423, (c) SEQ ID NO: 359, (d) SEQ ID NO: 380, (e) SEQID NO: 394 to 396 and (f) SEQ ID NO: 407 to 409,

4) combination of primers selected respectively one from (a) SEQ ID NO:424 to 427, (b) SEQ ID NO: 428 to 434, (c) SEQ ID NO: 360 to 361, (d)SEQ ID NO: 381 to 384, (e) SEQ ID NO: 397 to 398 and (f) SEQ ID NO: 411to 412;

32. A primer set for nucleic acid amplification for detecting humancytokeratin 19 comprising either one of the following groups 1) to3); 1) a primer set including oligonucleotides having base sequencesrepresented by SEQ ID NOs: 413, 418, 357 and 378 as primers,

2) a primer set including oligonucleotides having base sequencesrepresented by SEQ ID NOs: 419, 421, 358 and 379 as primers.

3) a primer set including oligonucleotides having base sequencesrepresented by SEQ ID NOs: 424, 431, 360 and 381 as primers;

33. A primer set for nucleic acid amplification for detecting humancytokeratin 19 comprising either one of the following groups 1) to4); 1) a primer set including oligonucleotides having base sequencesrepresented by SEQ ID NOs: 413, 418, 357, 378, 385 and 402 as primers,

2) a primer set including oligonucleotides having base sequencesrepresented by SEQ ID NOs: 419, 421, 358, 379, 392 and 404 as primers,

3) a primer set including oligonucleotides having base sequencesrepresented by SEQ ID NOs: 422, 423, 359, 380, 394 and 407 as primers,

4) a primer set including oligonucleotides having base sequencesrepresented by SEQ ID NOs: 424, 431, 360, 381, 397 and 411 as primers;

34. A method for detecting nucleic acids using a necessary primerselected from the primers according to the above 19. or 20., or a primerset selected from the primer sets according to any one of the above 21.to 33.;

35. A primer for nucleic acid amplification for detecting humancytokeratin 20 comprising an oligonucleotide having a sequence selectedfrom the following groups 1) to 5);

1) an oligonucleotide selected from a region of base position 340-490 ora region of base position 495-1050 in a base sequence of SEQ ID NO: 435and regions of a complementary strand thereof, containing at least 5 ormore successive bases in SEQ ID NO.435 and/or in the complementarystrand thereof,

2) an oligonucleotide having a base sequence represented by any one ofSEQ ID NOs: 436 to 460,

3) a complementary strand of the oligonucleotide according to theabove 1) or 2),

4) an oligonucleotide capable of hybridizing with the oligonucleotideaccording to any one of the above 1) to 3) under stringent conditions,and

5) an oligonucleotide comprising abase sequence obtainable from theoligonucleotides according to the above 1) to 4) wherein one to severalbases are replaced, inserted, added or otherwise mutated, theoligonucleotide having a primer function;

36. A primer for nucleic acid amplification for detecting humancytokeratin 20, comprising an oligonucleotide selected from the basesequences of SEQ ID NOs: 443, 444 or 454 to 474;

37. The primer for nucleic acid amplification for detecting humancytokeratin 20 according to the above 35. or 36, wherein the nucleicacid amplification is conducted by the LAMP method;

38. A primer set for nucleic acid amplification for detecting humancytokeratin 20, wherein at least two kinds of primers are selected fromprimers for nucleic acid amplification, each primer comprising anoligonucleotide comprising a sequence selected from the followinggroups 1) to 5);

1) an oligonucleotide selected from a region of base position 340-1050in a base sequence of SEQ ID NO: 435 and a complementary strand regionthereof, containing at least 5 or more successive bases in SEQ ID NO.435 and/or in the complementary strand thereof,

2) an oligonucleotide having a base sequence represented by any one ofSEQ ID NOs: 436 to 460,

3) a complementary strand of the oligonucleotide according to theabove 1) or 2),

4) an oligonucleotide capable of hybridizing with the oligonucleotideaccording to any one of the above 1) to 3) under stringent conditions,and

5) an oligonucleotide comprising a base sequence obtainable from theoligonucleotides according to the above 1) to 4) wherein one to severalbases are replaced, inserted, added or otherwise mutated, theoligonucleotide having a primer function;

39. The primer set for nucleic acid amplification for detecting humancytokeratin 20 according to the above 38., wherein the nucleic acidamplification is conducted by the LAMP method;

40. The primer set for nucleic acid amplification for detecting humancytokeratin 20 according to the above 39., wherein at least four kindsof primers are selected from the primers for nucleic acid amplificationeach comprising an oligonucleotide;

41. The primer set for detecting human cytokeratin 20 according to anyone of the above 38. to 40., wherein at least two kinds of primersincluded in the primer set respectively recognize two regions of thebase sequence of SEQ ID NO: 435 and/or a complementary strand thereof;

42. The primer set for detecting human cytokeratin 20 according to anyone of the above 38. to 41., wherein the primers included in said primerset recognize at least six regions in the base sequence represented bySEQ ID NO:435 and/or a complementary strand thereof;

43. A primer set comprising a combination of primers, one of the primersbeing selected from (a) SEQ ID NOs: 461 to 466, the other selected from(b) SEQ ID NOs: 468 to 473, among the primers for nucleic acidamplification for detecting human cytokeratin 20 each comprising anoligonucleotide selected from the base sequences of SEQ ID NOs: 461 to466 or SEQ ID NOs: 468 to 473;

44. The primer set for detecting human cytokeratin 20 according to theabove 43., further comprising oligonucleotides having base sequencesrepresented by SEQ ID NO: 444 and SEQ ID NO: 455 as primers;

45. The primer set for detecting human cytokeratin 20 according to theabove 43. or 44., further comprising an oligonucleotide having a basesequence represented by SEQ ID NO: 457 and/or 459 or 460 as a primer;

46. A primer set for detecting human cytokeratin 20 comprisingoligonucleotides having base sequences represented by SEQ ID NOs: 443,454, 467 and 474 as primers;

47. A primer set for detecting human cytokeratin 20, wherein anoligonucleotide having abase sequence represented by SEQ ID NO: 456and/or 458 is used as a primer in addition to oligonucleotides havingbase sequences represented by SEQ ID NOs: 443, 454, 467 and 474;

48. A method for detecting nucleic acids using a necessary primerselected from the primers according to the above 35. or 36., or a primerset selected from the primer sets according to any one of the above 37.to 47.;

49. The method for detecting nucleic acids according to the above 18.,34. or 48., wherein the nucleic acid amplification is conducted by theLAMP method;

50. A reagent for use in the method for detecting nucleic acidsaccording to the above 18., 34., 48. or 49.;

51. A reagent kit for use in the method for detecting nucleic acidsaccording to the above 18., 34., 48. or 49.; and

52. A nucleic acid detecting system using the method for detectingnucleic acids according to the above 18., 34., 48. or 49.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a view showing results of LAMP conducted by using Primer set Iof human CK18 (Test example 1-1);

FIG. 2 is a view showing results of LAMP conducted by using Primer setII of human CK18 (Test example 1-1);

FIG. 3 is a view showing results of LAMP conducted by using Primer setIII of human CK18 (Test example 1-1);

FIG. 4 is a view showing results of LAMP conducted by using Primer setIV of human CK18 (Test example 1-1);

FIG. 5 is a view showing results of LAMP conducted by using Primer set I(with loop primers) of human CK18 (Test example 1-2);

FIG. 6 is a view showing results of LAMP conducted by using Primer set I(without loop primers) of human CK18 (Test example 1-2);

FIG. 7 shows amplification specificity when LAMP is conducted usingPrimer set I of human CK18 (Test example 1-3);

FIG. 8 is a view showing results of LAMP conducted by using Primer set A(without loop primers) of human CK19 (Test example 2-1);

FIG. 9 is a view showing results of LAMP conducted by using Primer set A(without loop primers) of human CK19 (Test example 2-1);

FIG. 10 is a view showing results of LAMP conducted by using Primer setA (with loop primers) of human CK19 (Test example 2-1);

FIG. 11 is a view showing results of LAMP conducted by using Primer setA (with loop primers) of human CK19 (Test example 2-1);

FIG. 12 is a view showing results of LAMP conducted by using Primer setA (with loop primers) of human CK19 (Test example 2-1);

FIG. 13 is a view showing results of LAMP conducted by using Primer setC (without loop primers) of human CK19 (Test example 2-2);

FIG. 14 is a view showing results of LAMP conducted by using Primer setC (with loop primers) of human CK19 (Test example 2-2);

FIG. 15 is a view showing results of LAMP conducted by using Primer setC (with loop primers) of human CK19 (Test example 2-2);

FIG. 16 is a view showing results of LAMP conducted by using Primer setC (with loop primers) of human CK19 (Test example 2-2);

FIG. 17 is a view showing results of LAMP conducted by using Primer setC (with loop primers) of human CK19 (Test example 2-2);

FIG. 18 is a view showing results of LAMP conducted by using Primer setC (with loop primers) of human CK19 (Test example 2-2);

FIG. 19 is a view showing results of LAMP conducted by using Primer setC (with loop primers) of human CK19 (Test example 2-2);

FIG. 20 is a view showing results of LAMP conducted by using Primer setD (without loop primers) of human CK19 (Test example 2-3);

FIG. 21 is a view showing results of LAMP conducted by using Primer setD (with loop primers) of human CK19 (Test example 2-3);

FIG. 22 is a view showing results of LAMP conducted by using Primer setA of human CK19 (Test example 2-4);

FIG. 23 is a view showing results of LAMP conducted by using Primer setB of human CK19 (Test example 2-4);

FIG. 24 is a view showing results of LAMP conducted by using Primer setC of human CK19 (Test example 2-4);

FIG. 25 is a view showing results of LAMP conducted by using Primer setD of human CK19 (Test example 2-4);

FIG. 26 shows amplification specificity when LAMP is conducted usingPrimer set A of human CK19 (Test example 2-5);

FIG. 27 shows amplification specificity when LAMP is conducted usingPrimer set B of human CK19 (Test example 2-5);

FIG. 28 shows amplification specificity when LAMP is conducted usingPrimer set C of human CK19 (Test example 2-5);

FIG. 29 shows amplification specificity when LAMP is conducted usingPrimer set D of human CK19 (Test example 2-5);

FIG. 30 is a view showing results of LAMP conducted by using Primer setC of human CK19 (Test example 2-6);

FIG. 31 is a view showing results of LAMP conducted by using Primer setC of human CK19 (Test example 2-7);

FIG. 32 is a view showing results of LAMP conducted by using Primer set1 of human CK20 (Test example 3-2);

FIG. 33 shows amplification specificity when LAMP is conducted usingPrimer set 1 of human CK20 (Test example 3-3);

FIG. 34 is a view showing results of LAMP conducted by using Primer set3 of human CK20 (Test example 3-4);

FIG. 35 shows amplification specificity when LAMP is conducted usingPrimer set 3 of human CK20 (Test example 3-5);

BEST MODE FOR CARRYING OUT THE INVENTION

(Primer Designing)

The present invention provides a primer for nucleic acid amplificationapplicable to methods of amplifying nucleic acids of human CKs,preferably to the LAMP method. For example, the primer can be designedby selecting an appropriate oligonucleotide comprising at least 5 ormore successive bases in known CK base sequences represented by, forexample, SEQ ID NOs: 1, 342, 435 and/or complementary sequences thereof.

The basic concept of primers used in the LAMP method is as described inPatent document 1. Concretely, defining three regions F3c, F2c, F1c inthis order from 3′ end and three regions R3, R2, R1 in this order from5′ end of a target DNA to be amplified, and at least four primers aredesigned by selecting oligonucleotide chains comprising base sequencesthat are substantially identical with and/or substantially complementarywith those of at least the above six regions.

The wording “substantially identical base sequence” is defined asfollows. When a complementary strand that has been synthesized from atemplate having a certain base sequence hybridizes with a target basesequence and gives a starting point of synthesis of a complementarystrand, the base sequence is referred to be substantially identical withthe target base sequence. For example, the wording “base sequencesubstantially identically with F2” comprehends not only the basesequence that is perfectly identical with F2 but also the base sequencesserving as templates that hybridize with F2 and give a base sequenceserving as a starting point for synthesis of a complementary strand.

The term “identical” or “complementary” used herein for characterizing abase sequence constituting an oligonucleotide according to the presentinvention does not necessarily mean “perfectly identical” or “perfectlycomplementary”. In other words, when referring to “identical to acertain sequence”, sequences which are complementary to those capable ofhybridizing with the certain sequence are also included. On the otherhand, the term “complementary” means a sequence that is capable ofhybridizing under stringent conditions and providing 3′ end serving as astarting point of synthesis of a complementary strand.

Primers of the present invention have such a chain length that allowsbase-pair biding with a complementary strand while keeping a necessaryspecificity under a given environment in a variety of nucleic acidsynthesis reaction as will be described below. Concretely, they have alength of 5 to 200 bases, more preferably 10 to 50 bases. Since thechain length of a primer that can be recognized by a known polymerasecatalyzing sequence-dependent nucleic acid synthesis reaction is atleast around 5 bases, the length of the hybridizing part should belonger than that. Additionally, from the view point of keepingspecificity of a particular base sequence, the base sequence preferablyhas a length of not less than 10 bases. Too long base sequences aredifficult to be prepared by chemical synthesis, so that theaforementioned chain lengths are exemplified as a preferred range.

The term “template” used herein means a nucleic acid which serves as atemplate for synthesis of a complementary strand. A complementary strandwhose base sequence is complementary to a template is also interpretedas a strand capable of hybridizing with a template, however the relationbetween template and complementary strand is relative. In other words, acomplementary strand can become a template.

In the present invention, primers selected from a base sequence of atarget DNA each constitute either of FIP (forward inner primer), F3primer (forward outer primer), RIP (reverse inner primer) and R3 primer(reverse outer primer).

FIP is designed to have a base sequence of F2 region which issubstantially complementary with F2c region of a target DNA at its 3′end, and a base sequence which is substantially identical to F1c regionof the target DNA at its 5′ end. In this case, a sequence that does notdepend on the target DNA may exist between the sequences of F2 and F1c.The sequence that does not depend on the target DNA may have a length of0 to 50 bases, preferably 0 to 40 bases.

F3 primer is designed to have a base sequence substantially identicalwith F3 region which is substantially complementary with F3c region of atarget DNA.

RIP is designed to have a base sequence of R2 region which issubstantially complementary with R2c region of a target DNA at its 3′end, and a base sequence which is substantially identical to R1c regionof the target DNA at its 5′ end. Likewise FIP, a sequence that does notdepend on the target DNA may exist between the sequences of R2 and R1cin RIP.

R3 primer is designed to have a base sequence substantially identicalwith R3 region which is substantially complementary with R3c region of atarget DNA.

In the LAMP method, by additionally using at least one kind of loopprimer, it is possible to reduce the time for amplification (refer toInternational Publication No. WO02/24902). The term “loop primer” refersto a primer having a complementary sequence in a single-strand part ofthe 5′ end loop of the dumbbell structure, more concretely, between R1region and R2 region or between F1 region and F2 region, for example.Use of a loop primer makes it possible to increase the starting pointsof DNA synthesis. This loop primer is designed so as to allowhybridization in a loop region where FIP or RIP that is generated in thecourse of DNA synthesis will not hybridize.

(Designing of Primer for Detecting Human CK18)

A primer for detecting human CK18 is designed by selecting anappropriate oligonucleotide comprising at least 5 or more successivebases from a known base sequence of 1412 bases represented by SEQ ID NO:1 and/or a complementary sequence thereof. The base sequence representedby SEQ ID NO: 1 is based on Genbank accession No. 4557887.

A primer for detecting human CK18 is also designed by selecting a regionin accordance with the aforementioned principle of primer.

Taking care of the base composition, GC content, secondary structure, Tmvalue and the like, the region of a primer for detecting human CK18 isselected so that the base sequence recognizing a DNA region has a lengthof at least 5 bases, preferably 10 to 30 bases, more preferably 17 to 25bases. Tm value can be generally determined by Nearest Neighbor method.DNA region can be selected so that Tm value is 55 to 65° C., preferably58 to 64° C., and GC content is 40 to 70%, preferably 50 to 65%.

With such a condition, the region of primer selected in the presentinvention is included in a region of base position 270-1375 of the basesequence represented by SEQ ID NO:1 and a region of complementary strandthereof, and preferably in a region of base position 280-580 and aregion of complementary strand thereof.

A primer for detecting human cytokeratin CK18 is designed by selectingfrom the following groups 1) to 5); 1) an oligonucleotide included in aregion of base position 270-1375 in a base sequence of SEQ ID NO: 1and/or a complementary strand region thereof, containing at least 5bases; 2) an oligonucleotide having a base sequence represented by anyone of SEQ ID NOs: 2 to 41; 3) a complementary strand of theoligonucleotide according to the above 1) or 2); 4) an oligonucleotidecapable of hybridizing with the oligonucleotide according to any one ofthe above 1) to 3) under stringent conditions, and 5) an oligonucleotidecomprising abase sequence obtainable from the oligonucleotides accordingto the above 1) to 4) wherein one to several bases are replaced,inserted, added or otherwise mutated.

Oligonucleotides can be produced by a well-known method, for example bychemical synthesis. Alternatively, naturally occurring nucleic acid maybe digested with a restriction enzyme or the like, followed bymodification or coupling, thereby forming the base sequence as describedabove. Concretely, they can be synthesized using an oligonucleotidesynthesizer (Expedite Model 8909 DNA synthesizer, manufactured byApplied Biosystems) or the like. Also oligonucleotides in which one toseveral bases are replaced, inserted, added or otherwise mutated can besynthesized by a well-known process. For example, synthesis of sucholigonucleotide can be achieved by using site-directed mutagenesis, genehomologous recombination, primer extension or PCR technique singly or inappropriate combination, according to the method described in, forexample, Molecular Cloning: A Laboratory Manual (2nd edition, 1989)edited by Sambrook, et al., Cold Spring Harbor Laboratory Press., ColdSpring Harbor NY; Labomanual Genetic Engineering edited by MasakiMuramatsu, Maruzen, 1988; and PCR technology—Principal and applicationof DNA amplification 0edited by Ehrlichm HE., Stockton Press, 1989, ormodified methods of the above using, for example Ulmer's technique(Science (1983) 219:666).

As a stringent hybridization condition, generally known conditions canbe selected. One exemplary condition includes: overnight hybridizationat 42° C. in a solution containing 50% formamide, 5×SSC (150 mM NaCl, 15mM trisodium citrate), 50 mM sodium phosphate, pH 7.6, 5× Denhart'ssolution, 10% dextran sulfate, and 20 μg/ml of DNA; primary washing in2×SSC·0.1% SDS at room temperature; and secondary washing in0.1×SSC·0.1% SDS at 65° C.

In this case, since the nucleic acid template to be amplified is mRNA ofhuman CK18, primers to be used should be designed so as not to amplifygenomic DNA contained in the test sample. Concretely, at least one ofthe primers included in a primer set of the present invention preferablyincludes a region spanning a plurality of exons in human CK18 gene. Suchmeasure makes it possible to selectively amplify sequences from mRNA ofhuman CK18 while excluding amplification of sequences from genomic DNA.

(Designing of Primer for Detecting Human CK19)

Also a primer for detecting human CK19 is designed in a similar manneras a primer for detecting human CK18. A primer for detecting human CK19is designed by selecting an appropriate oligonucleotide comprising atleast 5 or more successive bases from a known base sequence of 1360bases represented by SEQ ID NO: 342 and/or a complementary sequencethereof. The base sequence represented by SEQ ID NO: 342 is based onGenbank accession No. 4504916.

Also a primer for detecting human CK19 is also designed by selecting aregion in accordance with the aforementioned principle of primer.

The region of primer for detecting human CK19 is included in a region ofbase position 270-930 of the base sequence represented by SEQ ID NO: 342and/or a region of complementary strand thereof, and preferably in aregion of base position 270-560, 370-585, 625-854 or 655-930 and/or aregion of complementary strand thereof.

A primer for detecting human CK19 is designed by selecting from thefollowing groups 1) to 5); 1) an oligonucleotide included in a region ofbase position 270-930 in a base sequence of SEQ ID NO: 342 and acomplementary strand region thereof, preferably included in a region ofbase position 270-560, 370-585, 625-854 or 655-930 and/or a region ofcomplementary strand thereof, containing at least 5 bases; 2) anoligonucleotide having a base sequence represented by any one of SEQ IDNOs: 343 to 382; 3) a complementary strand of the oligonucleotideaccording to the above 1) or 2); 4) an oligonucleotide capable ofhybridizing with the oligonucleotide according to any one of theabove 1) to 3) under stringent conditions, and 5) an oligonucleotidecomprising a base sequence obtainable from the oligonucleotidesaccording to the above 1) to 4) wherein one to several bases arereplaced, inserted, added or otherwise mutated.

(Designing of Primer for Detecting Human CK20)

Also a primer for detecting human CK20 is designed in a similar manneras a primer for detecting human CK18 or CK19. A primer for detectinghuman CK20 is designed by selecting an appropriate oligonucleotidecomprising at least 5 or more successive bases from a known basesequence of 1275 bases represented by SEQ ID NO: 435 and/or acomplementary sequence thereof. The base sequence represented by SEQ IDNO: 435 is based on Genbank accession No. 402644.

Also a primer for detecting human CK20 is also designed by selecting aregion in accordance with the aforementioned principle of primer.

The region of primer for detecting human CK20 is included in a region ofbase position 340-1050 of the base sequence represented by SEQ ID NO:435and a region of complementary strand thereof, preferably in a region ofbase position 340-490 or 495-1050 and a region of complementary strandthereof, and more preferably in a region of base position 340-490,495-570 or 790-1050 and a region of complementary strand thereof.

A primer for detecting human CK20 is designed by selecting from thefollowing groups 1) to 5); 1) an oligonucleotide included in a region ofbase position 340-1050 in a base sequence of SEQ ID NO: 435 and/or acomplementary strand region thereof, preferably included in a region ofbase position 340-490 or 495-1050 and/or a region of complementarystrand thereof, and more preferably included in a region of baseposition 340-490, 495-570 or 790-1050 and/or a region of complementarystrand thereof, containing at least 5 bases; 2) an oligonucleotidehaving a base sequence represented by any one of SEQ ID NOs: 436 to 474;3) a complementary strand of the oligonucleotide according to theabove 1) or 2); 4) an oligonucleotide capable of hybridizing with theoligonucleotide according to any one of the above 1) to 3) understringent conditions, and 5) an oligonucleotide comprising a basesequence obtainable from the oligonucleotides according to the above 1)to 4) wherein one to several bases are replaced, inserted, added orotherwise mutated.

(Primer Set)

In conducting amplification of nucleic acid using a primer of thepresent invention, combination of two or more kinds of primers are usedas a primer set. In the LAMP method, combination of at lest four kindsof primers (FIP, F3 primer, RIP, R3 primer) is used as a primer set.Additionally, one or more kind of loop primer may be combined in theprimer set.

(RT-LAMP Method)

RT-LAMP method is a kind of LAMP method which uses RNA as a template.Basic concept of the LAMP method is as described in InternationalPublication No. WO00/28082. In the RT-LAMP method, a starting pointstructure of LAMP method is synthesized while cDNA is synthesized from atemplate RNA in the same solution. More specifically, after thefollowing step 1), the steps of 2) to 5) are repeated for extension ofDNA, whereby amplification of target DNA is achieved.

1) FIP binds to a template RNA strand, and a complementary DNA strand tothe template RNA strand extends. For this reaction, reversetranscriptase such as reverse transcriptase from AMV is used.

2) A complementary DNA to the template RNA strand extends while the DNAstrand from FIP synthesized in the step 1) is peeled off the templateRNA by F3 primer. Extension of DNA strand after that time proceeds byvirtue of DNA polymerase.

3) RIP binds to the DNA strand peeled off in the step 2), and a DNAstrand extends.

4) A complementary DNA strand to the DNA from FIP extends, while R3primer peels off the DNA strand from RIP extended in the step 3),whereby a starting point structure of LAMP method is synthesized.

5) Since both ends of the DNA strand peeled off in the step 4) havesequences which are complementary with each other in the same DNAstrand, these sequences hybridize with each other, providing loopstructures on both ends.

Enzymes having both the reverse transcriptase activity and DNApolymerase activity such as BcaDNA polymerase are also known. By usingsuch an enzyme, the above reaction can be conducted with one enzyme.

(Detection Method)

A DNA strand synthesized in the LAMP method has a sequence which iscomplementary to the own sequence, so that most part of the DNA strandforms base-pair binding. Using this characteristic, it is possible todetect an amplified product. By conducting nucleic acid amplificationusing primers of the present invention in the presence of fluorochromessuch as ethidium bromide, SYBER GREEN I and Pico Green, which are doublestrand intercalators, increase in fluorescence intensity is observed asthe product increases. By monitoring this, it is possible tosimultaneously trace amplification of DNA and increase in fluorescencein a closed system (See “Kanai's manual of clinical laboratory medicine”the 31st ed., pp. 1318; JP-A 2001-242169, hereinafter simply referred toas “real-time method”).

Furthermore, in the LAMP method, insoluble magnesium pyrophosphate isgenerated as a by-product during the amplification reaction, resultingin white turbidness. Therefore, by detecting the turbidity by checkingthe turbidness of the reaction solution by eyes or by measuringabsorbance or scattered light intensity of the reaction solution, or byexamining the residue on a color filter after filtration of the reactionsolution through the filter, it is possible to determinepresence/absence of the amplified product (See International PublicationNo. WO01/83817).

(Reagents, Reagent Kit Etc.)

A variety of reagents required for detecting nucleic acids using primersof the present invention can be packed into a kit form in advance.Concretely, a variety of oligonucleotides required as primers forsynthesis of complementary strand or primers for replacement of thepresent invention; an enzyme having reverse transcriptase activity;dNTPs serving as substrates for synthesis of complementary strand; a DNApolymerase for conducting strand displacing synthesis of complementarystrand; a buffer for providing a desired condition for the enzymaticreaction; agents for removing substances that inhibit the amplificationreaction such as RNase inhibitor as necessary; and further reagentsrequired for detecting a reaction product as necessary are provided inthe form of a kit.

The present invention covers a primer and a primer set for nucleic acidamplification; a nucleic acid detecting method using these primers; adetection reagent used in the nucleic acid detecting method; a nucleicacid detecting kit; and an entire nucleic acid detecting system.

EXAMPLES

In the following, the present invention will be described in more detailby way of examples, however it is to be noted that the present inventionwill not be limited to these examples.

Example 1-1 Selection of Region from Human CK18 Base Sequence

The base sequence of human CK18 represented by SEQ ID NO: 1 was searchedfor positions of appropriate regions for the LAMP method using a probedesigning software. As a result of selecting regions according to thecriteria that Tm 58.5-63.5° C. for F1c and R1c, Tm 61.5-62.5° C. for F2and R2, Tm 58.5-62.5° C. for F3 and R3, the regions as shown below areselected. The selected regions are included in a region of base position340-1050 of the base sequence represented by SEQ ID NO: 1 and acomplementary strand region thereof.

F1c: Complementary strand region for base position on base sequencerepresented by SEQ ID NO: 1 442-420 tgaagtaatggctccagtctctg (SEQ ID NO:2) 439-418 agtaatggctccagtctctgac (SEQ ID NO: 3) 443-421ttgaagtaatggctccagtctct (SEQ ID NO: 4) 419-403 acctggggtcccttctt (SEQ IDNO: 5) 414-396 gggtcccttcttctccaag (SEQ ID NO: 6) 413-394ggtcccttcttctccaagtg (SEQ ID NO: 7) 412-393 gtcccttcttctccaagtgc (SEQ IDNO: 8) 411-392 tcccttcttctccaagtgct (SEQ ID NO: 9) 410-392cccttcttctccaagtgctc (SEQ ID NO: 10) 409-390 ccttcttctccaagtgctcc (SEQID NO: 11) 584-568 acagactggcgcatggc (SEQ ID NO: 12) 620-602tcaatgaccttgcggagcc (SEQ ID NO: 13) 621-602 atcaatgaccttgcggagcc (SEQ IDNO: 14) 622-603 catcaatgaccttgcggagc (SEQ ID NO: 15) 623-603tcatcaatgaccttgcggagc (SEQ ID NO: 16) 666-648 agcctcgatctctgtctcc (SEQID NO: 17) 743-726 gagctggcaatctgggct (SEQ ID NO: 18) 742-725agctggcaatctgggctt (SEQ ID NO: 19) 741-724 gctggcaatctgggcttg (SEQ IDNO: 20) 740-720 ctggcaatctgggcttgtagg (SEQ ID NO: 21) 739-720tggcaatctgggcttgtagg (SEQ ID NO: 22) 738-719 ggcaatctgggcttgtaggc (SEQID NO: 23) 756-740 cacggtcaacccagagc (SEQ ID NO: 24) 795-777gatcttggcgaggtcctga (SEQ ID NO: 25) 809-789 cggatgtctgccatgatcttg (SEQID NO: 26) 829-810 ccagctcgtcatattgggcc (SEQ ID NO: 27) 913-894cagcagactgtgtggtgacc (SEQ ID NO: 28) 941-924 gtgagcgtcgtctcagca (SEQ IDNO: 29) 1008-987  gctggccttcagatttctcatg (SEQ ID NO: 30) 355-335ccaggctcctcactctgtcca (SEQ ID NO: 31) 430-410 tccagtctctgacctggggtc (SEQID NO: 32) 588-569 ctccacagactggcgcatgg (SEQ ID NO: 33) 769-748gggcatctacctccacggtcaa (SEQ ID NO: 34) 897-877 gaccactgtggtgctctcctc(SEQ ID NO: 35) 925-904 cagctccaacctcagcagactg (SEQ ID NO: 36) 1263-1242ggtttgcatggagttgctgctg (SEQ ID NO: 37)

F2: Region of base position on base sequence represented by SEQ ID NO: 1376-392 gagagcaaaatccggga (SEQ ID NO: 38) 377-393 agagcaaaatccgggag (SEQID NO: 39) 378-394 gagcaaaatccgggagc (SEQ ID NO: 40) 384-400aatccgggagcacttgg (SEQ ID NO: 41) 369-385 gaggctggagagcaaaa (SEQ ID NO:42) 523-540 cgtcttgctgctgatgac (SEQ ID NO: 43) 524-542gtcttgctgctgatgactt (SEQ ID NO: 44) 543-565 tagagtcaagtatgagacagagc (SEQID NO: 45) 544-565 agagtcaagtatgagacagagc (SEQ ID NO: 46) 546-566agtcaagtatgagacagagct (SEQ ID NO: 47) 588-604 gaacgacatccatgggc (SEQ IDNO: 48) 660-676 cgaggctctcaaggagg (SEQ ID NO: 49) 661-677gaggctctcaaggagga (SEQ ID NO: 50) 662-678 aggctctcaaggaggag (SEQ ID NO:51) 687-706 catgaagaagaaccacgaag (SEQ ID NO: 52) 719-736gcctacaagcccagattg (SEQ ID NO: 53) 747-763 gttgaccgtggaggtag (SEQ ID NO:54) 768-784 ccccaaatctcaggacc (SEQ ID NO: 55) 839-855 accgagaggagctagac(SEQ ID NO: 56) 878-894 aggagagcaccacagtg (SEQ ID NO: 57) 943-960gagctgagacgtacagtc (SEQ ID NO: 58) 295-314 gagaccatgcaaagcctgaa (SEQ IDNO: 59) 369-388 gaggctggagagcaaaatcc (SEQ ID NO: 60) 523-542cgtcttgctgctgatgactt (SEQ ID NO: 61) 708-729 ggaagtaaaaggcctacaagcc (SEQID NO: 62) 837-857 gaaccgagaggagctagacaa (SEQ ID NO: 63) 864-884gtctcagcagattgaggagag (SEQ ID NO: 64) 1202-1221 tggaagatggcgaggacttt(SEQ ID NO: 65)

F3: Region of base position on base sequence represented by SEQ ID NO: 1322-338 ctggcctcttacctgga (SEQ ID NO: 66) 293-309 aggagaccatgcaaagc (SEQID NO: 67) 470-489 tcttcgcaaatactgtggac (SEQ ID NO: 68) 466-486cagatcttcgcaaatactgtg (SEQ ID NO: 69) 473-491 tcgcaaatactgtggacaa (SEQID NO: 70) 476-495 caaatactgtggacaatgcc (SEQ ID NO: 71) 523-540cgtcttgctgctgatgac (SEQ ID NO: 72) 546-566 agtcaagtatgagacagagct (SEQ IDNO: 73) 624-643 caccaatatcacacgactgc (SEQ ID NO: 74) 687-706catgaagaagaaccacgaag (SEQ ID NO: 75) 695-712 agaaccacgaagaggaag (SEQ IDNO: 76) 747-763 gttgaccgtggaggtag (SEQ ID NO: 77) 812-829cccaatatgacgagctgg (SEQ ID NO: 78) 845-864 aggagctagacaagtactgg (SEQ IDNO: 79) 855-873 caagtactggtctcagcag (SEQ ID NO: 80) 907-923tctgctgaggttggagc (SEQ ID NO: 81) 275-294 gaggcatccagaacgagaag (SEQ IDNO: 82) 349-366 agcctggagaccgagaac (SEQ ID NO: 83) 490-507aatgcccgcatcgttctg (SEQ ID NO: 84) 672-690 ggaggagctgctcttcatg (SEQ IDNO: 85) 807-824 ccgggcccaatatgacga (SEQ ID NO: 86) 840-859ccgagaggagctagacaagt (SEQ ID NO: 87) 1176-1193 tgagatcgccacctaccg (SEQID NO: 88)

R1c: Region of base position on base sequence represented by SEQ ID NO:1 444-463 gatcatcgaggacctgaggg (SEQ ID NO: 89) 420-442cagagactggagccattacttca (SEQ ID NO: 90) 424-447 gactggagccattacttcaagatc(SEQ ID NO: 91) 425-448 actggagccattacttcaagatca (SEQ ID NO: 92) 426-450ctggagccattacttcaagatcatc (SEQ ID NO: 93) 427-451tggagccattacttcaagatcatcg (SEQ ID NO: 94) 428-451ggagccattacttcaagatcatcg (SEQ ID NO: 95) 429-453gagccattacttcaagatcatcgag (SEQ ID NO: 96) 430-454agccattacttcaagatcatcgagg (SEQ ID NO: 97) 431-454gccattacttcaagatcatcgagg (SEQ ID NO: 98) 432-456ccattacttcaagatcatcgaggac (SEQ ID NO: 99) 433-457cattacttcaagatcatcgaggacc (SEQ ID NO: 100) 587-605 agaacgacatccatgggct(SEQ ID NO: 101) 588-606 gaacgacatccatgggctc (SEQ ID NO: 102) 589-607aacgacatccatgggctcc (SEQ ID NO: 103) 590-607 acgacatccatgggctcc (SEQ IDNO: 104) 598-614 catgggctccgcaaggt (SEQ ID NO: 105) 632-649tcacacgactgcagctgg (SEQ ID NO: 106) 624-645 caccaatatcacacgactgcag (SEQID NO: 107) 630-649 tatcacacgactgcagctgg (SEQ ID NO: 108) 631-649atcacacgactgcagctgg (SEQ ID NO: 109) 685-708 ttcatgaagaagaaccacgaagag(SEQ ID NO: 110) 739-756 agctctgggttgaccgtg (SEQ ID NO: 111) 740-756gctctgggttgaccgtg (SEQ ID NO: 112) 741-757 ctctgggttgaccgtgg (SEQ ID NO:113) 742-758 tctgggttgaccgtgga (SEQ ID NO: 114) 743-759ctgggttgaccgtggag (SEQ ID NO: 115) 744-760 tgggttgaccgtggagg (SEQ ID NO:116) 746-764 ggttgaccgtggaggtaga (SEQ ID NO: 117) 747-767gttgaccgtggaggtagatgc (SEQ ID NO: 118) 748-767 ttgaccgtggaggtagatgc (SEQID NO: 119) 749-767 tgaccgtggaggtagatgc (SEQ ID NO: 120) 750-768gaccgtggaggtagatgcc (SEQ ID NO: 121) 751-768 accgtggaggtagatgcc (SEQ IDNO: 122) 766-783 gcccccaaatctcaggac (SEQ ID NO: 123) 812-831cccaatatgacgagctggct (SEQ ID NO: 124) 855-877 caagtactggtctcagcagattg(SEQ ID NO: 125) 924-941 tgctgagacgacgctcac (SEQ ID NO: 126) 947-966tgagacgtacagtccagtcc (SEQ ID NO: 127) 1016-1032 acagcctgagggaggtg (SEQID NO: 128) 360-379 cgagaaccggaggctggaga (SEQ ID NO: 129) 443-464agatcatcgaggacctgagggc (SEQ ID NO: 130) 592-611 gacatccatgggctccgcaa(SEQ ID NO: 131) 778-799 caggacctcgccaagatcatgg (SEQ ID NO: 132) 900-921cacacagtctgctgaggttgga (SEQ ID NO: 133) 928-948 gagacgacgctcacagagctg(SEQ ID NO: 134) 1277-1296 ccacccgccggatagtggat (SEQ ID NO: 135)

R2: Complementary strand region for base position on base sequencerepresented by SEQ ID NO: 1 540-523 gtcatcagcagcaagacg (SEQ ID NO: 136)541-523 agtcatcagcagcaagacg (SEQ ID NO: 137) 494-475gcattgtccacagtatttgc (SEQ ID NO: 138) 493-474 cattgtccacagtatttgcg (SEQID NO: 139) 492-473 attgtccacagtatttgcga (SEQ ID NO: 140) 491-473ttgtccacagtatttgcga (SEQ ID NO: 141) 490-472 tgtccacagtatttgcgaa (SEQ IDNO: 142) 489-470 gtccacagtatttgcgaaga (SEQ ID NO: 143) 488-468tccacagtatttgcgaagatc (SEQ ID NO: 144) 487-467 ccacagtatttgcgaagatct(SEQ ID NO: 145) 486-466 cacagtatttgcgaagatctg (SEQ ID NO: 146) 678-662ctcctccttgagagcct (SEQ ID NO: 147) 677-661 tcctccttgagagcctc (SEQ ID NO:148) 676-660 cctccttgagagcctcg (SEQ ID NO: 149) 675-659ctccttgagagcctcga (SEQ ID NO: 150) 673-657 ccttgagagcctcgatc (SEQ ID NO:151) 672-655 cttgagagcctcgatctc (SEQ ID NO: 152) 667-651gagcctcgatctctgtc (SEQ ID NO: 153) 666-649 agcctcgatctctgtctc (SEQ IDNO: 154) 665-649 gcctcgatctctgtctc (SEQ ID NO: 155) 713-696acttcctcttcgtggttc (SEQ ID NO: 156) 721-702 ggccttttacttcctcttcg (SEQ IDNO: 157) 714-696 tacttcctcttcgtggttc (SEQ ID NO: 158) 762-746tacctccacggtcaacc (SEQ ID NO: 159) 809-792 cggatgtctgccatgatc (SEQ IDNO: 160) 808-790 ggatgtctgccatgatctt (SEQ ID NO: 161) 829-812ccagctcgtcatattggg (SEQ ID NO: 162) 877-858 caatctgctgagaccagtac (SEQ IDNO: 163) 874-856 tctgctgagaccagtactt (SEQ ID NO: 164) 922-906ctccaacctcagcagac (SEQ ID NO: 165) 985-969 agtccaggtcgatctcc (SEQ ID NO:166) 1006-987  tggccttcagatttctcatg (SEQ ID NO: 167) 1011-994 caagctggccttcagatt (SEQ ID NO: 168) 1086-1070 aaggtgcagcaggatcc (SEQ IDNO: 169) 437-417 taatggctccagtctctgacc (SEQ ID NO: 170) 515-496tcaatctgcagaacgatgcg (SEQ ID NO: 171) 669-649 gagagcctcgatctctgtctc (SEQID NO: 172) 669-650 gagagcctcgatctctgtct (SEQ ID NO: 173) 857-837ttgtctagctcctctcggttc (SEQ ID NO: 174) 857-838 ttgtctagctcctctcggtt (SEQID NO: 175) 981-962 caggtcgatctccaaggact (SEQ ID NO: 176) 1008-989 gctggccttcagatttctca (SEQ ID NO: 177) 1350-1331 gctggcttaatgcctcagaa(SEQ ID NO: 178)

R3: Complementary strand region for base position on base sequencerepresented by SEQ ID NO: 1 566-546 agctctgtctcatacttgact (SEQ ID NO:179) 541-523 agtcatcagcagcaagacg (SEQ ID NO: 180) 540-5239gtcatcagcagcaagacg (SEQ ID NO: 181) 721-702 ggccttttacttcctcttcg (SEQID NO: 182) 713-696 acttcctcttcgtggttc (SEQ ID NO: 183) 740-724ctggcaatctgggcttg (SEQ ID NO: 184) 786-769 gaggtcctgagatttggg (SEQ IDNO: 185) 854-837 tctagctcctctcggttc (SEQ ID NO: 186) 877-858caatctgctgagaccagtac (SEQ ID NO: 187) 922-906 ctccaacctcagcagac (SEQ IDNO: 188) 910-894 cagactgtgtggtgacc (SEQ ID NO: 189) 893-877actgtggtgctctcctc (SEQ ID NO: 190) 960-943 gactgtacgtctcagctc (SEQ IDNO: 191) 1021-1005 ggctgttctccaagctg (SEQ ID NO: 192) 1056-1039catctgtagggcgtagcg (SEQ ID NO: 193) 1141-1125 catactcctgggcctgg (SEQ IDNO: 194) 476-458 gcgaagatctgagccctca (SEQ ID NO: 195) 538-521catcagcagcaagacggg (SEQ ID NO: 196) 688-670 tgaagagcagctcctcctt (SEQ IDNO: 197) 885-866 gctctcctcaatctgctgag (SEQ ID NO: 198) 1008-989 gctggccttcagatttctca (SEQ ID NO: 199) 1030-1012 cctccctcaggctgttctc (SEQID NO: 200) 1370-1352 ccaaagggtaccctgcttc (SEQ ID NO: 201)

Loop F: Complementary strand region for base position on base sequencerepresented by SEQ ID NO: 1 419-403 acctggggtcccttctt (SEQ ID NO: 202)414-396 gggtcccttcttctccaag (SEQ ID NO: 203) 413-394ggtcccttcttctccaagtg (SEQ ID NO: 204) 399-380 caagtgctcccggattttgc (SEQID NO: 205) 398-380 aagtgctcccggattttgc (SEQ ID NO: 206) 397-379agtgctcccggattttgct (SEQ ID NO: 207) 396-378 gtgctcccggattttgctc (SEQ IDNO: 208) 395-377 tgctcccggattttgctct (SEQ ID NO: 209) 394-376gctcccggattttgctctc (SEQ ID NO: 210) 567-544 cagctctgtctcatacttgactct(SEQ ID NO: 211) 584-568 acagactggcgcatggc (SEQ ID NO: 212) 709-688cctcttcgtggttcttcttcat (SEQ ID NO: 213) 916-898 cctcagcagactgtgtggt (SEQID NO: 214) 913-894 cagcagactgtgtggtgacc (SEQ ID NO: 215) 567-544cagctctgtctcatacttgactct (SEQ ID NO: 216) 743-726 gagctggcaatctgggct(SEQ ID NO: 217) 1243-1224 tgtccaaggcatcaccaaga (SEQ ID NO: 218)1242-1222 gtccaaggcatcaccaagatt (SEQ ID NO: 219)

Loop R: Region of base position on base sequence represented by SEQ IDNO: 1 474-495 cgcaaatactgtggacaatgcc (SEQ ID NO: 220) 466-489cagatcttcgcaaatactgtggac (SEQ ID NO: 221) 444-463 gatcatcgaggacctgaggg(SEQ ID NO: 222) 626-646 ccaatatcacacgactgcagc (SEQ ID NO: 223) 617-640ttgatgacaccaatatcacacgac (SEQ ID NO: 224) 632-649 tcacacgactgcagctgg(SEQ ID NO: 225) 659-676 tcgaggctctcaaggagg (SEQ ID NO: 226) 767-784cccccaaatctcaggacc (SEQ ID NO: 227) 970-986 gagatcgacctggactc (SEQ IDNO: 228) 622-643 gacaccaatatcacacgactgc (SEQ ID NO: 229) 621-643tgacaccaatatcacacgactgc (SEQ ID NO: 230) 623-644 acaccaatatcacacgactgca(SEQ ID NO: 231) 810-829 ggcccaatatgacgagctgg (SEQ ID NO: 232) 1296-1315tggcaaagtggtgtctgaga (SEQ ID NO: 233)

Example 1-2 Designing of Primer for Detecting CK18

From the sequences of the regions selected in Example 1-1, the followingprimers for nucleic acid amplification to be applied to the LAMP methodwere obtained.

FIP: Primer having base sequence in which base sequences of regions F1cand F2 are coupled 09FA971-376 tgaagtaatggctccagtctctggagagcaaaatccggga(SEQ ID NO: 234) 09FA971-377 tgaagtaatggctccagtctctgagagcaaaatccgggag(SEQ ID NO: 235) 09FA971-378 tgaagtaatggctccagtctctggagcaaaatccgggagc(SEQ ID NO: 236) 09FA971-384 tgaagtaatggctccagtctctgaatccgggagcacttgg(SEQ ID NO: 237) 09FA974-376 agtaatggctccagtctctgacgagagcaaaatccggga(SEQ ID NO: 238) 09FA974-377 agtaatggctccagtctctgacagagcaaaatccgggag(SEQ ID NO: 239) 09FA974-378 agtaatggctccagtctctgacgagcaaaatccgggagc(SEQ ID NO: 240) 09FA974-384 agtaatggctccagtctctgacaatccgggagcacttgg(SEQ ID NO: 241) 09FA970-376 ttgaagtaatggctccagtctctgagagcaaaatccggga(SEQ ID NO: 242) 09FA970-377 ttgaagtaatggctccagtctctagagcaaaatccgggag(SEQ ID NO: 243) 09FA970-378 ttgaagtaatggctccagtctctgagcaaaatccgggagc(SEQ ID NO: 244) 09FA970-384 ttgaagtaatggctccagtctctaatccgggagcacttgg(SEQ ID NO: 245) 09FA999-369 gggtcccttcttctccaaggaggctggagagcaaaa (SEQID NO: 246) 09FA994-369 acctggggtcccttcttgaggctggagagcaaaa (SEQ ID NO:247) 09FA1000-369 ggtcccttcttctccaagtggaggctggagagcaaaa (SEQ ID NO: 248)09FA1002-369 tcccttcttctccaagtgctgaggctggagagcaaaa (SEQ ID NO: 249)09FA1004-369 ccttcttctccaagtgctccgaggctggagagcaaaa (SEQ ID NO: 250)12FA829-523 acagactggcgcatggccgtcttgctgctgatgac (SEQ ID NO: 251)12FA829-524 acagactggcgcatggcgtcttgctgctgatgactt (SEQ ID NO: 252)13FA793-543 tcaatgaccttgcggagcctagagtcaagtatgagacagagc (SEQ ID NO: 253)13FA793-544 tcaatgaccttgcggagccagagtcaagtatgagacagagc (SEQ ID NO: 254)13FA793-546 tcaatgaccttgcggagccagtcaagtatgagacagagct (SEQ ID NO: 255)13FA792-543 atcaatgaccttgcggagcctagagtcaagtatgagacagagc (SEQ ID NO: 256)13FA792-544 atcaatgaccttgcggagccagagtcaagtatgagacagagc (SEQ ID NO: 257)13FA792-546 atcaatgaccttgcggagccagtcaagtatgagacagagct (SEQ ID NO: 258)13FA791-543 catcaatgaccttgcggagctagagtcaagtatgagacagagc (SEQ ID NO: 259)13FA791-544 catcaatgaccttgcggagcagagtcaagtatgagacagagc (SEQ ID NO: 260)13FA791-546 catcaatgaccttgcggagcagtcaagtatgagacagagct (SEQ ID NO: 261)13FA790-543 tcatcaatgaccttgcggagctagagtcaagtatgagacagagc (SEQ ID NO:262) 13FA790-544 tcatcaatgaccttgcggagcagagtcaagtatgagacagagc (SEQ ID NO:263) 13FA790-546 tcatcaatgaccttgcggagcagtcaagtatgagacagagct (SEQ ID NO:264) 14FA747-588 agcctcgatctctgtctccgaacgacatccatgggc (SEQ ID NO: 265)18FA675-660 ggcaatctgggcttgtaggccgaggctctcaaggagg (SEQ ID NO: 266)18FA670-660 gagctggcaatctgggctcgaggctctcaaggagg (SEQ ID NO: 267)18FA670-662 gagctggcaatctgggctaggctctcaaggaggag (SEQ ID NO: 268)18FA674-660 tggcaatctgggcttgtaggcgaggctctcaaggagg (SEQ ID NO: 269)18FA674-662 tggcaatctgggcttgtaggaggctctcaaggaggag (SEQ ID NO: 270)18FA675-661 ggcaatctgggcttgtaggcgaggctctcaaggagga (SEQ ID NO: 271)18FA675-662 ggcaatctgggcttgtaggcaggctctcaaggaggag (SEQ ID NO: 272)19FA657-687 cacggtcaacccagagccatgaagaagaaccacgaag (SEQ ID NO: 273)21FA618-719 gatcttggcgaggtcctgagcctacaagcccagattg (SEQ ID NO: 274)21FA604-747 cggatgtctgccatgatcttggttgaccgtggaggtag (SEQ ID NO: 275)23FA584-768 ccagctcgtcatattgggccccccaaatctcaggacc (SEQ ID NO: 276)27FA500-839 cagcagactgtgtggtgaccaccgagaggagctagac (SEQ ID NO: 277)29FA472-878 gtgagcgtcgtctcagcaaggagagcaccacagtg (SEQ ID NO: 278)32FA405-943 gctggccttcagatttctcatggagctgagacgtacagtc (SEQ ID NO: 279) ek335-295 ccaggctcctcactctgtccagagaccatgcaaagcctgaa (SEQ ID NO: 280) ek410-369 tccagtctctgacctggggtcgaggctggagagcaaaa (SEQ ID NO: 281) ek569-523 ctccacagactggcgcatggcgtcttgctgctgatgac (SEQ ID NO: 282) ek748-708 gggcatctacctccacggtcaaggaagtaaaaggcctacaagcc (SEQ ID NO: 283) ek877-837 gaccactgtggtgctctcctcgaaccgagaggagctagacaa (SEQ ID NO: 284) ek904-864 cagctccaacctcagcagactggtctcagcagattgaggagag (SEQ ID NO: 285) ek1242-1202 ggtttgcatggagttgctgctgtggaagatggcgaggacttt (SEQ ID NO: 286)RIP: Primer having base sequence in which base sequences of regions R1cand R2 are coupled 09RA444-873 gatcatcgaggacctgaggggtcatcagcagcaagacg(SEQ ID NO: 287) 09RA444-872 gatcatcgaggacctgagggagtcatcagcagcaagacg(SEQ ID NO: 288) 09RA420-927cagagactggagccattacttcacacagtatttgcgaagatctg (SEQ ID NO: 289)09RA420-925 cagagactggagccattacttcatccacagtatttgcgaagatc (SEQ ID NO:290) 09RA420-923 cagagactggagccattacttcatgtccacagtatttgcgaa (SEQ ID NO:291) 09RA420-921 cagagactggagccattacttcaattgtccacagtatttgcga (SEQ ID NO:292) 09RA420-919 cagagactggagccattacttcagcattgtccacagtatttgc (SEQ ID NO:293) 09RA424-927 gactggagccattacttcaagatccacagtatttgcgaagatctg (SEQ IDNO: 294) 09RA424-923 gactggagccattacttcaagatctgtccacagtatttgcgaa (SEQ IDNO: 295) 09RA424-921 gactggagccattacttcaagatcattgtccacagtatttgcga (SEQID NO: 296) 12RA598-737 catgggctccgcaaggtcctccttgagagcctcg (SEQ ID NO:297) 12RA587-746 agaacgacatccatgggctgagcctcgatctctgtc (SEQ ID NO: 298)12RA588-737 gaacgacatccatgggctccctccttgagagcctcg (SEQ ID NO: 299)12RA588-746 gaacgacatccatgggctcgagcctcgatctctgtc (SEQ ID NO: 300)12RA588-748 gaacgacatccatgggctcgcctcgatctctgtctc (SEQ ID NO: 301)12RA590-737 acgacatccatgggctcccctccttgagagcctcg (SEQ ID NO: 302)12RA590-746 acgacatccatgggctccgagcctcgatctctgtc (SEQ ID NO: 303)12RA590-748 acgacatccatgggctccgcctcgatctctgtctc (SEQ ID NO: 304)12RA598-740 catgggctccgcaaggtccttgagagcctcgatc (SEQ ID NO: 305)12RA598-746 catgggctccgcaaggtgagcctcgatctctgtc (SEQ ID NO: 306)13RA632-700 tcacacgactgcagctggacttcctcttcgtggttc (SEQ ID NO: 307)13RA632-692 tcacacgactgcagctggggccttttacttcctcttcg (SEQ ID NO: 308)13RA632-699 tcacacgactgcagctggtacttcctcttcgtggttc (SEQ ID NO: 309)13RA624-700 caccaatatcacacgactgcagacttcctcttcgtggttc (SEQ ID NO: 310)13RA624-699 caccaatatcacacgactgcagtacttcctcttcgtggttc (SEQ ID NO: 311)13RA624-692 caccaatatcacacgactgcagggccttttacttcctcttcg (SEQ ID NO: 312)13RA631-700 atcacacgactgcagctggacttcctcttcgtggttc (SEQ ID NO: 313)13RA631-699 atcacacgactgcagctggtacttcctcttcgtggttc (SEQ ID NO: 314)13RA631-692 atcacacgactgcagctggggccttttacttcctcttcg (SEQ ID NO: 315)13RA630-700 tatcacacgactgcagctggacttcctcttcgtggttc (SEQ ID NO: 316)13RA630-699 tatcacacgactgcagctggtacttcctcttcgtggttc (SEQ ID NO: 317)13RA630-692 tatcacacgactgcagctggggccttttacttcctcttcg (SEQ ID NO: 318)14RA685-651 ttcatgaagaagaaccacgaagagtacctccacggtcaacc (SEQ ID NO: 319)18RA743-604 ctgggttgaccgtggagcggatgtctgccatgatc (SEQ ID NO: 320)18RA743-605 ctgggttgaccgtggagggatgtctgccatgatctt (SEQ ID NO: 321)18RA747-604 gttgaccgtggaggtagatgccggatgtctgccatgatc (SEQ ID NO: 322)18RA749-604 tgaccgtggaggtagatgccggatgtctgccatgatc (SEQ ID NO: 323)18RA751-604 accgtggaggtagatgcccggatgtctgccatgatc (SEQ ID NO: 324)18RA749-605 tgaccgtggaggtagatgcggatgtctgccatgatctt (SEQ ID NO: 325)18RA751-605 accgtggaggtagatgccggatgtctgccatgatctt (SEQ ID NO: 326)19RA766-584 gcccccaaatctcaggacccagctcgtcatattggg (SEQ ID NO: 327)21RA812-536 cccaatatgacgagctggctcaatctgctgagaccagtac (SEQ ID NO: 328)23RA855-491 caagtactggtctcagcagattgctccaacctcagcagac (SEQ ID NO: 329)27RA924-428 tgctgagacgacgctcacagtccaggtcgatctcc (SEQ ID NO: 330)29RA947-402 tgagacgtacagtccagtcccaagctggccttcagatt (SEQ ID NO: 331)29RA947-407 tgagacgtacagtccagtcctggccttcagatttctcatg (SEQ ID NO: 332)32RA1016-327 acagcctgagggaggtgaaggtgcagcaggatcc (SEQ ID NO: 333) ek360-417 cgagaaccggaggctggagataatggctccagtctctgacc (SEQ ID NO: 334) ek443-496 agatcatcgaggacctgagggctcaatctgcagaacgatgcg (SEQ ID NO: 335) ek592-649 gacatccatgggctccgcaagagagcctcgatctctgtctc (SEQ ID NO: 336) ek592-650 gacatccatgggctccgcaagagagcctcgatctctgtct (SEQ ID NO: 337) ek778-837 caggacctcgccaagatcatgggaaccgagaggagctagacaa (SEQ ID NO: 338) ek900-962 cacacagtctgctgaggttggacaggtcgatctccaaggact (SEQ ID NO: 339) ek928-989 gagacgacgctcacagagctggctggccttcagatttctca (SEQ ID NO: 340) ek1277-1331 ccacccgccggatagtggatgctggcttaatgcctcagaa (SEQ ID NO: 341)F3 primer: (primer comprising base sequence represented by respectiveSEQ ID NO)

F309-322 (SEQ ID NO: 66), F309-293 (SEQ ID NO: 67), F312-470 (SEQ ID NO:68), F312-466 (SEQ ID NO: 69), F312-473 (SEQ ID NO: 70), F312-476 (SEQID NO: 71), F313-523 (SEQ ID NO: 72), F314-546 (SEQ ID NO: 73), F319-624(SEQ ID NO: 74), F321-687 (SEQ ID NO: 75), F321-695 (SEQ ID NO: 76),F323-747 (SEQ ID NO: 77), F327-812 (SEQ ID NO: 78), F329-845 (SEQ ID NO:79), F329-855 (SEQ ID NO: 80), F332-907 (SEQ ID NO: 81), F3 8 (SEQ IDNO: 82), F3 13 (SEQ ID NO: 83), F3 14 (SEQ ID NO: 84), F3 16 (SEQ ID NO:85), F3 23 (SEQ ID NO: 86), F3 29 (SEQ ID NO: 87) F3 37 (SEQ ID NO: 88)

R3 primer: (primer comprising base sequence represented by respectiveSEQ ID NO)

R309-847 (SEQ ID NO: 179), R309-872 (SEQ ID NO: 180), R309-873 (SEQ IDNO: 181), R312-692 (SEQ ID NO: 182), R312-700 (SEQ ID NO: 183),R313-673(SEQ ID NO: 184), R314-627 (SEQ ID NO: 185), R318-559(SEQ ID NO:186), R319-536 (SEQ ID NO: 187), R321-491(SEQ ID NO: 188), R321-503 (SEQID NO: 189), R321-520(SEQ ID NO: 190), R323-453 (SEQ ID NO: 191),R327-392(SEQ ID NO: 192), R329-357 (SEQ ID NO: 193), R332-272(SEQ ID NO:194), B3 8 (SEQ ID NO: 195), B3 13 (SEQ ID NO: 196), B3 14 (SEQ ID NO:197), B3 19 (SEQ ID NO: 198), B3 21 (SEQ ID NO: 199), B3 35 (SEQ ID NO:200), B3 37 (SEQ ID NO: 201)

Loop primer: (primer comprising base sequence represented by respectiveSEQ ID NO)

LF09-994 (SEQ ID NO: 202), LF09-999 (SEQ ID NO: 203), LF09-1000 (SEQ IDNO: 204), LF09-1014(SEQ ID NO: 205), LF09-1015 (SEQ ID NO: 206),LF09-1016(SEQ ID NO: 207), LF09-1017 (SEQ ID NO: 208), LF09-1018(SEQ IDNO: 209), LF09-1019 (SEQ ID NO: 210), LF12-846 (SEQ ID NO: 211),LF13-829 (SEQ ID NO: 212), LF18-704 (SEQ ID NO: 213), LF29-497 (SEQ IDNO: 214), LF29-500 (SEQ ID NO: 215), LF 14 (SEQ ID NO: 216), LF 20 (SEQID NO: 217), LF 371 (SEQ ID NO: 218), LF 372 (SEQ ID NO: 219) LR09-474(SEQ ID NO: 220), LR09-466 (SEQ ID NO: 221), LR09-444 (SEQ ID NO: 222),LR12-626 (SEQ ID NO: 223), LR12-617 (SEQ ID NO: 224), LR12-632 (SEQ IDNO: 225), LR13-659 (SEQ ID NO: 226), LR18-767 (SEQ ID NO: 227), LR29-970(SEQ ID NO: 228), LB 14 (SEQ ID NO: 229), LB 151 (SEQ ID NO: 230), LB152 (SEQ ID NO: 231), LB 371 (SEQ ID NO: 232), LB 372 (SEQ ID NO: 233)

Example 2-1 Selection of Region from Human CK19 Base Sequence

The base sequence of human CK19 represented by SEQ ID NO: 342 wassearched for positions of appropriate regions for the LAMP method usinga probe designing software. As a result of selecting regions accordingto the criteria that Tm 58.5-63.5° C. for F1c and R1c, Tm 61.5-62.5° C.for F2 and R2, Tm 58.5-62.5° C. for F3 and R3, the regions as shownbelow are selected. The selected regions are included in a region ofbase position 270-930 of the base sequence represented by SEQ ID NO: 342and a complementary strand region thereof.

F1c: Regions on complementary strand of base sequence represented by SEQID NO: 342 426-405 5′-tgtagtagtggctgtagtcgcg-3′ (SEQ ID NO: 343) 429-4075′-tcgtgtagtagtggctgtagtcg-3′ (SEQ ID NO: 344) 479-4585′-ggagttctcaatggtggcacca-3′ (SEQ ID NO: 345) 716-7005′-ttggcccctcagcgtac-3′ (SEQ ID NO: 346) 752-7355′-agcggaatccacctccac-3′ (SEQ ID NO: 347) 747-7285′-aatccacctccacactgacc-3′ (SEQ ID NO: 348) 746-7285′-atccacctccacactgacc-3′ (SEQ ID NO: 349) 745-7285′-tccacctccacactgacc-3′ (SEQ ID NO: 350)

F2: Regions on base sequence represented by SEQ ID NO: 342 352-3705′-agctagaggtgaagatccg-3′ (SEQ ID NO: 351) 364-3805′-agatccgcgactggtac-3′ (SEQ ID NO: 352) 360-376 5′-gtgaagatccgcgactg-3′(SEQ ID NO: 353) 417-437 5′-actactacacgaccatccagg-3′ (SEQ ID NO: 354)658-674 5′-aagagctggcctacctg-3′ (SEQ ID NO: 355) 690-7095′-gaggaaatcagtacgctgag-3′ (SEQ ID NO: 356)

F3: Regions on base sequence represented by SEQ ID NO: 342 275-2935′-gctaaccatgcagaacctc-3′ (SEQ ID NO: 357) 375-3925′-tggtaccagaagcagggg-3′ (SEQ ID NO: 358) 628-6455′-acctggagatgcagatcg-3′ (SEQ ID NO: 359) 658-6745′-aagagctggcctacctg-3′ (SEQ ID NO: 360) 661-677 5′-agctggcctacctgaag-3′(SEQ ID NO: 361)

R1c: Regions on base sequence represented by SEQ ID NO: 342 533-5165′-gtgccaccattgagaactcc-3′ (SEQ ID NO: 362) 485-5055′-tgtcctgcagatcgacaacgc-3′ (SEQ ID NO: 363) 486-5065′-gtcctgcagatcgacaacgcc-3′ (SEQ ID NO: 364) 727-7445′-aggtcagtgtggaggtgg-3′ (SEQ ID NO: 365) 764-7835′-tctcgccaagatcctgagtg-3′ (SEQ ID NO: 366) 766-7855′-tcgccaagatcctgagtgac-3′ (SEQ ID NO: 367) 772-7935′-agatcctgagtgacatgcgaag-3′ (SEQ ID NO: 368)

R2: Regions on complementary strand of base sequence represented by SEQID NO: 342 533-516 5′-ggttcggaagtcatctgc-3′ (SEQ ID NO: 369) 545-5265′-cgtctcaaacttggttcgga-3′ (SEQ ID NO: 370) 547-5285′-tccgtctcaaacttggttcg-3′ (SEQ ID NO: 371) 790-7735′-gcatgtcactcaggatc-3′ (SEQ ID NO: 372) 841-8245′-caggcttcagcatccttc-3′ (SEQ ID NO: 373) 848-8325′-ggtgaaccaggcttcag-3′ (SEQ ID NO: 374) 847-831 5′-gtgaaccaggcttcagc-3′(SEQ ID NO: 375) 845-828 5′-gaaccaggcttcagcatc-3′ (SEQ ID NO: 376)843-827 5′-accaggcttcagcatcc-3′ (SEQ ID NO: 377)

R3: Regions on complementary strand of base sequence represented by SEQID NO: 342 556-540 5′-agagcctgttccgtctc-3′ (SEQ ID NO: 378) 567-5845′-gtggaggccgacatcaac-3′ (SEQ ID NO: 379) 841-8245′-caggcttcagcatccttc-3′ (SEQ ID NO: 380) 916-9005′-tcggacctgctcatctg-3′ (SEQ ID NO: 381) 925-9085′-tcagtaacctcggacctg-3′ (SEQ ID NO: 382) 923-9075′-agtaacctcggacctgc-3′ (SEQ ID NO: 383) 921-905 5′-taacctcggacctgctc-3′(SEQ ID NO: 384)

Loof F: Regions on complementary strand of base sequence represented bySEQ ID NO: 342 395-381 5′-aggcccctgcttctg-3′ (SEQ ID NO: 385) 393-3795′-gcccctgcttctggt-3′ (SEQ ID NO: 386) 392-376 5′-cccctgcttctggtacc-3′(SEQ ID NO: 387) 392-375 5′-cccctgcttctggtacca-3′ (SEQ ID NO: 388)393-376 5′-gcccctgcttctggtacc-3′ (SEQ ID NO: 389) 395-3805′-aggcccctgcttctgg-3′ (SEQ ID NO: 390) 394-379 5′-ggcccctgcttctggt-3′(SEQ ID NO: 391) 457-440 5′-agaatcttgtcccgcagg-3′ (SEQ ID NO: 392)456-440 5′-gaatcttgtcccgcagg-3′ (SEQ ID NO: 393) 699-6805′-tgatttcctcctcatggttc-3′ (SEQ ID NO: 394) 698-6795′-gatttcctcctcatggttct-3′ (SEQ ID NO: 395) 694-6765′-tcctcctcatggttcttct-3′ (SEQ ID NO: 396) 734-3185′-actgacctggcctccca-3′ (SEQ ID NO: 397) 724-710 5′-cctcccacttggccc-3′(SEQ ID NO: 398)

Loop R: Regions on complementary strand of base sequence represented bySEQ ID NO: 342 493-510 5′-agatcgacaacgcccgtc-3′ (SEQ ID NO: 399) 495-5125′-atcgacaacgcccgtctg-3′ (SEQ ID NO: 400) 495-509 5′-atcgacaacgcccgt-3′(SEQ ID NO: 401) 496-509 5′-tcgacaacgcccgt-3′ (SEQ ID NO: 402) 506-5205′-ccgtctggctgcaga-3′ (SEQ ID NO: 403) 507-520 5′-cgtctggctgcagatga-3′(SEQ ID NO: 404) 508-525 5′-gtctggctgcagatgact-3′ (SEQ ID NO: 405)509-526 5′-tctggctgcagatgactt-3′ (SEQ ID NO: 406) 752-7675′-tccgggcaccgatctc-3′ (SEQ ID NO: 407) 756-771 5′-ggcaccgatctcgcca-3′(SEQ ID NO: 408) 755-769 5′-gggcaccgatctcgc-3′ (SEQ ID NO: 409) 757-7715′-gcaccgatctcgcca-3′ (SEQ ID NO: 410) 805-822 5′-tcatggccgagcagaacc-3′(SEQ ID NO: 411) 806-821 5′-catggccgagcagaac-3′ (SEQ ID NO: 412)

Example 2-2 Designing of Primer for Detecting CK19

From the sequences of the regions selected in Example 2-1, the followingprimers for nucleic acid amplification to be applied to the LAMP methodwere obtained.

Each primer is shown in a primer set to be used in the RT-LAMP method ofthe present invention, and classified into four Groups A to D dependingon the region. Each primer belonging to Group A is selected from aregion of base position 270-560 in the base sequence represented by SEQID NO: 342 and a region of complementary strand thereof; likewise eachprimer belonging to Group B is selected from a region of base position370-585 and a region of complementary strand thereof; likewise eachprimer belonging to Group C is selected from a region of base position625-854 and a region of complementary strand thereof; and likewise eachprimer belonging to Group D is selected from a region of base position655-930 and a region of complementary strand thereof.

(Group A) FIP: Base Sequences of Regions F1c and F2 Coupled FA-4015′-tgtagtagtggctgtagtcgcgagctagaggtgaagatccg-3′ (SEQ ID NO: 413) (Basesequences of SEQ ID NO: 343 and SEQ ID NO: 351 are coupled) FA-4035′-tgtagtagtggctgtagtcgcgagatccgcgactggtac-3′ (SEQ ID NO: 414) (Basesequences of SEQ ID NO: 343 and SEQ ID NO: 352 are coupled) FA-4045′-tcgtgtagtagtggctgtagtcgagctagaggtgaagatccg-3′ (SEQ ID NO: 415) (Basesequences of SEQ ID NO: 344 and SEQ ID NO: 351 are coupled) FA-4055′-tcgtgtagtagtggctgtagtcggtgaagatccgcgactg-3′ (SEQ ID NO: 416) (Basesequences of SEQ ID NO: 344 and SEQ ID NO: 353 are coupled) FA-4065′-tcgtgtagtagtggctgtagtcgagatccgcgactggtac-3′ (SEQ ID NO: 417) (Basesequences of SEQ ID NO: 344 and SEQ ID NO: 352 are coupled) RIP: BaseSequences of Regions R1c and R2 Coupled RA-4015′-gtgccaccattgagaactccggttcggaagtcatctgc-3′ (SEQ ID NO: 418) (Basesequences of SEQ ID NO: 362 and SEQ ID NO: 369 are coupled) F3 primer:(identical sequence with base sequence of F3 region) F3-4015′-gctaaccatgcagaacctc-3′ (SEQ ID NO: 357) R3 primer: (identicalsequence with base sequence of R3 region) R3-401 5′-agagcctgttccgtctc-3′(SEQ ID NO: 378) Loop primer: (identical sequence with base sequence ofloop F region or loop R) LPF-401 5′-aggcccctgcttctg-3′ (SEQ ID NO: 385)LPF-402 5′-gcccctgcttctggt-3′ (SEQ ID NO: 386) LPF-4035′-cccctgcttctggtacc-3′ (SEQ ID NO: 387) LPF-4045′-cccctgcttctggtacca-3′ (SEQ ID NO: 388) LPF-4055′-gcccctgcttctggtacc-3′ (SEQ ID NO: 389) LPF-406 5′-aggcccctgcttctgg-3′(SEQ ID NO: 390) LPF-407 5′-ggcccctgcttctggt-3′ (SEQ ID NO: 391) LPR-4015′-agatcgacaacgcccgtc-3′ (SEQ ID NO: 399) LPR-4025′-atcgacaacgcccgtctg-3′ (SEQ ID NO: 400) LPR-403 5′-atcgacaacgcccgt-3′(SEQ ID NO: 401) LPR-404 5′-tcgacaacgcccgt-3′ (SEQ ID NO: 402)

(Group B) FIP: Base Sequences of Regions F1c and F2 Coupled FA1-EK5′-ggagttctcaatggtggcaccaactactacacgaccatccagg-3′ (SEQ ID NO: 419) (Basesequences of SEQ ID NO: 346 and SEQ ID NO: 354 are coupled) RIP: BaseSequences of Regions R1c and R2 Coupled RA2-EK5′-tgtcctgcagatcgacaacgccgtctcaaacttggttcgga-3′ (SEQ ID NO: 420) (Basesequences of SEQ ID NO: 363 and SEQ ID NO: 370 are coupled) RA6-EK5′-gtcctgcagatcgacaacgcctccgtctcaaacttggttcg-3′ (SEQ ID NO: 421) (Basesequences of SEQ ID NO: 364 and SEQ ID NO: 371 are coupled) F3 primer:(identical sequence with base sequence of F3 region) F3-EK5′-tggtaccagaagcagggg-3′ (SEQ ID NO: 358) R3 primer: (identical sequencewith base sequence of R3 region) R3-EK 5′-gtggaggccgacatcaac-3′ (SEQ IDNO: 379) Loop primer: (identical sequence with base sequence of loop Fregion or loop R) LPF1-EK 5′-agaatcttgtcccgcagg-3′ (SEQ ID NO: 392)LPF2-EK 5′-gaatcttgtcccgcagg-3′ (SEQ ID NO: 393) LPR1-EK5′-ccgtctggctgcaga-3′ (SEQ ID NO: 403) LPR2-EK 5′-cgtctggctgcagatga-3′(SEQ ID NO: 404) LPR3-EK 5′-gtctggctgcagatgact-3′ (SEQ ID NO: 405)LPR4-EK 5′-tctggctgcagatgactt-3′ (SEQ ID NO: 406)

(Group C) FIP: Base Sequences of Regions F1c and F2 Coupled FA-11015′-ttggcccctcagcgtacaagagctggcctacctg-3′ (SEQ ID NO: 422) (Basesequences of SEQ ID NO: 346 and SEQ ID NO: 355 are coupled) RIP: BaseSequences of Regions R1c and R2 Coupled RA-11015′-aggtcagtgtggaggtggcgcatgtcactcaggatc-3′ (SEQ ID NO: 423) (Basesequences of SEQ ID NO: 365 and SEQ ID NO: 372 are coupled) F3 primer:(identical sequence with base sequence of F3 region) F3-11015′-acctggagatgcagatcg-3′ (SEQ ID NO: 359) R3 primer: (identical sequencewith base sequence of R3 region) R3-1101 5′-caggcttcagcatccttc-3′ (SEQID NO: 380) Loop primer: (identical sequence with base sequence of loopF region or loop R) LPF-1100 5′-tgatttcctcctcatggttc-3′ (SEQ ID NO: 394)LPF-1102 5′-gatttcctcctcatggttct-3′ (SEQ ID NO: 395) LPF-11035′-tcctcctcatggttcttct-3′ (SEQ ID NO: 396) LPR-11015′-tccgggcaccgatctc-3′ (SEQ ID NO: 407) LPR-1102 5′-ggcaccgatctcgcca-3′(SEQ ID NO: 408) LPR-1103 5′-gggcaccgatctcgc-3′ (SEQ ID NO: 409)LPR-1104 5′-gcaccgatctcgcca-3′ (SEQ ID NO: 410)

(Group D) FIP: Base Sequences of Regions F1c and F2 Coupled FA-6015′-agcggaatccacctccacgaggaaatcagtacgctgag-3′ (SEQ ID NO: 424) (Basesequences of SEQ ID NO: 347 and SEQ ID NO: 356 are coupled) FA-6025′-aatccacctccacactgaccgaggaaatcagtacgctgag-3′ (SEQ ID NO: 425) (Basesequences of SEQ ID NO: 348 and SEQ ID NO: 356 are coupled) FA-6035′-atccacctccacactgaccgaggaaatcagtacgctgag-3′ (SEQ ID NO: 426) Basesequences of SEQ ID NO: 349 and SEQ ID NO: 359 are coupled) FA-6045′-tccacctccacactgaccgaggaaatcagtacgctgag-3′ (SEQ ID NO: 427) (Basesequences of SEQ ID NO: 350 and SEQ ID NO: 356 are coupled) RIP: BaseSequences of Regions R1c and R2 Coupled RA-6015′-tctcgccaagatcctgagtgcaggcttcagcatccttc-3′ (SEQ ID NO: 428) (Basesequences of SEQ ID NO: 366 and SEQ ID NO: 373 are coupled) RA-6025′-tctcgccaagatcctgagtgggtgaaccaggcttcag-3′ (SEQ ID NO: 429) (Basesequences of SEQ ID NO: 366 and SEQ ID NO: 374 are coupled) RA-6035′-tctcgccaagatcctgagtggtgaaccaggcttcagc-3′ (SEQ ID NO: 430) (Basesequences of SEQ ID NO: 366 and SEQ ID NO: 375 are coupled) RA-6045′-tctcgccaagatcctgagtggaaccaggcttcagcatc-3′ (SEQ ID NO: 431) (Basesequences of SEQ ID NO: 366 and SEQ ID NO: 376 are coupled) RA-6055′-tctcgccaagatcctgagtgaccaggcttcagcatcc-3′ (SEQ ID NO: 432) (Basesequences of SEQ ID NO: 366 and SEQ ID NO: 377 are coupled) RA-6065′-tcgccaagatcctgagtgaccaggcttcagcatccttc-3′ (SEQ ID NO: 433) (Basesequences of SEQ ID NO: 367 and SEQ ID NO: 373 are coupled) RA-6075′-agatcctgagtgacatgcgaagcaggcttcagcatccttc-3′ (SEQ ID NO: 434) (Basesequences of SEQ ID NO: 368 and SEQ ID NO: 373 are coupled) F3 primer:(identical sequence with base sequence of F3 region) F3-6015′-aagagctggcctacctg-3′ (SEQ ID NO: 360) F3-602 5′-agctggcctacctgaag-3′(SEQ ID NO: 361) R3 primer: (identical sequence with base sequence of R3region) R3-601 5′-tcggacctgctcatctg-3′ (SEQ ID NO: 381) R3-6025′-tcagtaacctcggacctg-3′ (SEQ ID NO: 382) R3-603 5′-agtaacctcggacctgc-3′(SEQ ID NO: 383) R3-604 5′-taacctcggacctgctc-3′ (SEQ ID NO: 384) Loopprimer: (identical sequence with base sequence of loop F region or loopR) LPF-601 5′-actgacctggcctccca-3′ (SEQ ID NO: 397) LPF-6025′-cctcccacttggccc-3′ (SEQ ID NO: 398) LPR-601 5′-tcatggccgagcagaacc-3′(SEQ ID NO: 411) LPR-602 5′-catggccgagcagaac-3′ (SEQ ID NO: 412)

Example 3-1 Selection of Region from Human CK20 Base Sequence

The base sequence of human CK20 represented by SEQ ID NO: 435 wassearched for positions of appropriate regions for the LAMP method usinga probe designing software. As a result of selecting regions accordingto the criteria that Tm 58.5-63.5° C. for F1c and R1c, Tm 61.5-62.5° C.for F2 and R2, Tm 58.5-62.5° C. for F3 and R3, the regions as shownbelow are selected. The selected regions are included in a region ofbase position 340-1050 of the base sequence represented by SEQ ID NO:435 and a complementary strand region thereof.

F1c: Regions on complementary strand of base sequence represented by SEQID NO: 435 920-900 5′-ttcatgctgagatgggactgg-3′ (SEQ ID NO: 436) 915-8955′-gctgagatgggactggagttc-3′ (SEQ ID NO: 437) 436-4165′-caatttgcaggacacaccgag-3′ (SEQ ID NO: 438)

F2: Regions on base sequence represented by SEQ ID NO: 435 847-8655′-gaggttcaactaacggagc-3′ (SEQ ID NO: 439) 850-8695′-gttcaactaacggagctgag-3′ (SEQ ID NO: 440) 855-8725′-actaacggagctgagacg-3′ (SEQ ID NO: 441) 370-3885′-attgaagagctgcgaagtc-3′ (SEQ ID NO: 442)

F3: Regions on base sequence represented by SEQ ID NO: 435 345-3675′-cgactacagtgcatattacagac-3′ (SEQ ID NO: 443) 805-8225′-cagcaacaggtcacagtg-3′ (SEQ ID NO: 444)

R1c: Regions on base sequence represented by SEQ ID NO: 435 940-9585′-ctagaggagaccaaggccc-3′ (SEQ ID NO: 445) 939-9585′-tctagaggagaccaaggccc-3′ (SEQ ID NO: 446) 947-9665′-agaccaaggcccgttacagc-3′ (SEQ ID NO: 447) 452-4725′-ctgctgaggacttcagactga-3′ (SEQ ID NO: 448)

R2: Regions on complementary strand of base sequence represented by SEQID NO: 435 1004-987  5′-agagagctcaacagcgac-3′ (SEQ ID NO: 449) 1007-990 5′-tccagagagctcaacagc-3′ (SEQ ID NO: 450)  994-9785′-acagcgactggaggttg-3′ (SEQ ID NO: 451) 1000-9845′-agctcaacagcgactgg-3′ (SEQ ID NO: 452)  523-5055′-cttggagatcagcttccac-3′ (SEQ ID NO: 453)

R3: Regions on complementary strand of base sequence represented by SEQID NO: 435  556-535 5′-gtagggttaggtcatcaaagac-3′ (SEQ ID NO: 454)1044-1027 5′-gcgttccatgttactccg-3′ (SEQ ID NO: 455)

Loof F: Regions on complementary strand of base sequence represented bySEQ ID NO: 435 409-389 5′-gcagttgagcatccttaatct-3′ (SEQ ID NO: 456)891-875 5′-ctcaaggctctgggagg-3′ (SEQ ID NO: 457)

Loop R: Regions on base sequence represented by SEQ ID NO: 435 480-4995′-gactgagagaggaatacgtc-3′ (SEQ ID NO: 458) 968-9855′-gccagttagccaacctcc-3′ (SEQ ID NO: 459) 970-985 5′-cagttagccaacctcc-3′(SEQ ID NO: 460)

Example 3-2 Designing of Primer for Detecting CK20

From the sequences of the regions selected in Example 3-1, the followingprimers for nucleic acid amplification to be applied to the LAMP methodwere obtained.

FIP: Base Sequences of Regions F1c and F2 Coupled KFA-55′-ttcatgctgagatgggactgggaggttcaactaacggagc-3′ (SEQ ID NO: 461) (Basesequences of SEQ ID NO: 436 and SEQ ID NO: 439 are coupled) KFA-5a5′-ttcatgctgagatgggactgggttcaactaacggagctgag-3′ (SEQ ID NO: 462) (Basesequences of SEQ ID NO: 436 and SEQ ID NO: 440 are coupled) KFA-5b5′-ttcatgctgagatgggactggactaacggagctgagacg-3′ (SEQ ID NO: 463) (Basesequences of SEQ ID NO: 436 and SEQ ID NO: 441 are coupled) KFA-5d5′-gctgagatgggactggagttcgaggttcaactaacggagc-3′ (SEQ ID NO: 464) (Basesequences of SEQ ID NO: 437 and SEQ ID NO: 439 are coupled) KFA-5e5′-gctgagatgggactggagttcgttcaactaacggagctgag-3′ (SEQ ID NO: 465) (Basesequences of SEQ ID NO: 437 and SEQ ID NO: 440 are coupled) KFA-5f5′-gctgagatgggactggagttcactaacggagctgagacg-3′ (SEQ ID NO: 466) (Basesequences of SEQ ID NO: 437 and SEQ ID NO: 441 are coupled) AFA5′-caatttgcaggacacaccgagattgaagagctgcgaagtc-3′ (SEQ ID NO: 467) (Basesequences of SEQ ID NO: 438 and SEQ ID NO: 442 are coupled) RIP: BaseSequences of Regions R1c and R2 Coupled

RIP: Base Sequences of Regions R1c and R2 Coupled KRA-55′-ctagaggagaccaaggcccagagagctcaacagcgac-3′ (SEQ ID NO: 468) (Basesequences of SEQ ID NO: 445 and SEQ ID NO: 449 are coupled) KRA-5a5′-tctagaggagaccaaggccctccagagagctcaacagc-3′ (SEQ ID NO: 469) (Basesequences of SEQ ID NO: 446 and SEQ ID NO: 450 are coupled) KRA-5c5′-tctagaggagaccaaggcccacagcgactggaggttg-3′ (SEQ ID NO: 470) (Basesequences of SEQ ID NO: 446 and SEQ ID NO: 451 are coupled) KRA-5d5′-agaccaaggcccgttacagcagagagctcaacagcgac-3′ (SEQ ID NO: 471) (Basesequences of SEQ ID NO: 447 and SEQ ID NO: 449 are coupled) KRA-5e5′-agaccaaggcccgttacagctccagagagctcaacagc-3′ (SEQ ID NO: 472) (Basesequences of SEQ ID NO: 447 and SEQ ID NO: 450 are coupled) KRA-5f5′-agac caaggcccgttacagcagctcaacagcgactgg-3′ (SEQ ID NO: 473) (Basesequences of SEQ ID NO: 447 and SEQ ID NO: 452 are coupled) ARAf5′-ctgctgaggacttcagactgacttggagatcagcttccac-3′ (SEQ ID NO: 474) (Basesequences of SEQ ID NO: 448 and SEQ ID NO: 453 are coupled)

F3 primer: (identical sequence with base sequence of F3 region) AF35′-cgactacagtgcatattacagac-3′ (SEQ ID NO: 443) KF3-55′-cagcaacaggtcacagtg-3′ (SEQ ID NO: 444)

R3 primer: (identical sequence with base sequence of R3 region) AR35′-gtagggttaggtcatcaaagac-3′ (SEQ ID NO: 454) KR3-55′-gcgttccatgttactccg-3′ (SEQ ID NO: 455)

Loop primer: (identical sequence with base sequence of loop F region orloop R) LPF2 5′-gcagttgagcatccttaatct-3′ (SEQ ID NO: 456) K-LPF25′-ctcaaggctctgggagg-3′ (SEQ ID NO: 457) LPR6 5′-gactgagagaggaatacgtc-3′(SEQ ID NO: 458) K-LPR1 5′-gccagttagccaacctcc-3′ (SEQ ID NO: 459) K-LPR25′-cagttagccaacctcc-3′ (SEQ ID NO: 460)

TEST EXAMPLES

Effects obtained when the RT-LAMP method is executed using differentprimers for detecting CK18, CK19 or CK20 obtained above were measuredand determined according to the Test examples 1 to 3.

Test Example 1-1 Observation of Amplification

This test was conducted in order to examine the time required fromstarting of the reaction until amplification could be observed in eachcase the measurement was conducted by RT-LAMP method using the followingcombination of various primers for detecting human CK18.

1) Method for Preparing Human CK18RNA Sample

By conducting RT-PCR using a primer designed based on a base sequence ofhuman CK18, human CK18cDNA was isolated from total RNA derived fromKATOIII (stomach cancer cell). From human CK18cDNA cloned intopBluescript (plasmid manufactured by STRATAGENE), a transcriptionproduct was synthesized using in vitro transcription system (Riboprobein vitro transcription system (manufactured by Promega)). RNAconcentration of the undiluted solution thus obtained was determined bymeasuring absorbance at 260 nm, and based on the result, the solutionwas diluted in 50 ng/μl yeast RNA (manufactured by Ambion) so that thecopy numbers of RNA of human CK18 were 60000, 6000, 600, 60, 6 and 0(control), which were used as template solutions.

2) Primer Set for Detecting Human CK18

A variety of primers were used in the combinations as shown in Table 1.TABLE 1 Primer set Primer set I II III IV FIP 234 252 259 278 RIP 287297 307 332 F3 primer 66 68 72 79 R3 primer 179 182 184 193 Loop primer203 211 212 214 (loop F) Loop primer 220 223 226 228 (loop R)(Each number represents SEQ ID number, and each primer means a primercomprising a sequence represented by each SEQ ID number.)

3) Composition of Reaction Solution dNTPs (manufactured by GIBCO) 0.4 mMMgSO₄ 2 mM Dithiothreitol 5 mM Betaine (manufactured by Sigma) 640 mMThermopol buffer (manufactured by New England BioLabs) AMV reversetranscriptase (manufactured by Promega) 1.25 U Bst DNA polymerase(manufactured by New 16 U England BioLabs) Ethidium bromide 0.125 mg/mLPrimer FIP 40 pmol, RIP 40 pmol, F3 primer  5 pmol, R3 primer  5 pmol,Loop primer (loop F, loop R) each 20 pmol4) RT-LAMP Method

To 23 μl of reaction solution containing the above six kinds of primers,2 μl of RNA sample of human CK18 was added, and heated at 65° C. for onehour.

5) Observation of Amplification

Since the amplified product has a double-strand structure, ethidiumbromide intercalates into the double-strand structure to emitfluorescent. Increase in fluorescence intensity (Rn) was measured inreal time by using PRISM 7700 manufactured by ABI.

6) Results

Results of the cases where Primer sets I to IV were respectively usedare shown in FIGS. 1 to 4. These results show that the larger the amountof the template of human CK18, the shorter the time required untilamplification can be observed in every set. In Primer sets I and III,amplification was observed in 20 minutes even in the case of 600 copies,while in Primer set II, amplification was observed after about 20minutes in the case of 6000 copies. As for Primer set IV, amplificationwas observed after about 20 minutes in the case of 6000 copies, andafter about 30 minutes even in the case of 600 copies.

Test Example 1-2 Effect of Loop Primer

Among Primer sets considered in Test example 1-1, Primer set I in whichthe time required for observation of amplification was shortest wasselected, and a test was made in order to examine the sensitivity inpresence/absence of a loop primer.

1) Method for Preparing Human CK18RNA Sample

Samples having copy numbers of 60000, 6000, 600 and 0 (control) wereprepared in a similar manner as described in Test example 1-1.

2) Primer Set

Primer set I shown in Table 1, and a primer set excluding each loopprimer from Primer set I were used.

3) Composition of Reaction Solution

A reaction solution similar to that of Test example 1-1 was used, and asto the set excluding loop primer, any loop primer was not added.

4) RT-LAMP Method

To 23 μl of reaction solution containing six or four kinds of primers, 2μl of RNA sample of human CK18 was added, and heated at 65° C. for onehour.

5) Observation of Amplification

Measurement was conducted in real time in a similar manner with Testexample 1-1.

6) Results

Results of determination are shown in FIGS. 5 and 6. These results showthat the time required until amplification can be observed was shorterin the case where a loop primer was not used. However, even in the casewhere a loop primer was used, amplification of human CK18 was observedafter about 50 minutes even in the case of 60000 copies.

Example 1-3 Amplification Specificity to Human CK18

Amplification specificity to human CK18 when measurement was conductedusing Primer set I was examined. It is known that cytokeratins (CK) haveisoforms such as human CK19, 20 and the like besides human CK18. Theseisoforms comprises a base sequence having about 60% homology with humanCK18. This test was conducted for determining whether human CK18 couldbe tested distinguishably from human CK19 or 20 when measurement wasconducted by using the above primer sets.

1) Method for Preparing RNA Sample

An RNA sample of human CK18 having a copy number of 60000 was preparedin a similar manner as described in Test example 1-1. Also with regardto RNAs of human CK19 and human CK20, samples were prepared in a similarmanner.

2) Primer Set

Primer set I shown in Table 1 was used.

3) Composition of Reaction Solution

A reaction solution similar to that of Test example 1-1 was used.

4) RT-LAMP Method

The similar condition as described in Test example 1 was used.

5) Observation of Amplification

Measurement was conducted in real time in a similar manner with Testexample 1-1.

6) Results

Results are shown in FIG. 7. These results show that human CK18RNA, butabsolutely not human CK19RNA and human CK20RNA are amplified when Primerset I was used, revealing specificity of Primer set I to human CK18RNA.

Test Example 2-1 Effect of Primer Sets (Group A) for Detecting HumanCK19

This test was conducted in order to examine an amplification patternwhen RNA of human CK19 was measured by the RT-LAMP method using a primerset selected from Group A shown in Example 2-2.

1) Primer Set (Group A)

Examination was carried out using a primer set consisting of FIP:FA-401, RIP: RA-401, F3: F3-401 and R3: R3-401, and a primer set incombination with various loop primers: LPF-401 and LPR-401, LPF-401 andLPR-402, LPF-401 and LPR-403, or LPF-401 and LPR-404.

2) Method for Preparing Human CK19RNA

By conducting RT-PCR using a primer designed based on a base sequence ofhuman CK19, human CK19cDNA was isolated from total RNA derived fromKATOIII (stomach cancer cell). From human CK19cDNA cloned intopBluescript (plasmid manufactured by STRATAGENE), a transcriptionproduct was synthesized using in vitro transcription system (Riboprobein vitro transcription system (manufactured by Promega)). RNAconcentration of the undiluted solution thus obtained was determined bymeasuring absorbance at 260 nm, and based on the result, the solutionwas diluted in 50 ng/μl yeast RNA (manufactured by Ambion) so that thecopy number of RNA of human CK19 was 60000, 6000, 600, 60, 6 and 0(control), which were used as template solutions.

3) Composition of Reaction Solution

A reaction solution similar to that of Test example 1-1 was used, and asto the set excluding loop primer, a loop primer was not added.

4) RT-LAMP Method

To 23 μl of reaction solution containing four kinds of primers notincluding loop primers or containing six kinds of primers including loopprimers, 2 μl of RNA sample of human CK19 was added, and heated at 65°C. for one hour.

5) Observation of Amplification

Measurement was conducted in real time in a similar manner with Testexample 1-1.

6) Results

Results obtained for the primer set not including loop primers and theprimer set including various loop primers are shown in FIGS. 8 to 12.These results show that the time required until amplification can beobserved was about 40 minutes after starting the test when loop primersare not used. On the other hand, as shown in FIGS. 9 to 12, in the caseof the systems including loop primers, amplification could be observedat about between 10 minutes and 20 minutes.

Test Example 2-2 Effect of Primer Sets (Group C) for Detecting HumanCK19

This test was conducted in order to examine an amplification patternwhen RNA of human CK19 was measured by the RT-LAMP method using a primerset selected from Group A shown in Example 2-2.

1) Primer Set (Group C)

Examination was carried out using a primer set consisting of FIP:FA-1101, RIP: RA-1101, F3: F3-1101 and R3: R3-1101, and a primer set incombination with various loop primers: LPF-1101 and LPR-1101, LPF-1101and LPR-1102, LPF-1101 and LPR-1103, LPF-1101 and LPR-1104, LPF-1102 andLPR-1101, or LPF-1103 and LPR-1101.

2) Preparation of Human CK19RNA Sample

Following the operation as shown in Test example 2-1, samples wereprepared so that the copy numbers were 60000, 6000, 600, 60 and 0(control), respectively.

3) Composition of Reaction Solution, 4) RT-LAMP Method and 5)Observation of Amplification were Conducted in a Similar Manner as TestExample 2-1.

6) Results

Results obtained for the primer set not including loop primers and theprimer set including various loop primers are shown in FIGS. 13 to 19.These results show that when loop primers are not used, the timerequired until amplification can be observed was about 30 minutes afterstarting the test (FIG. 13). On the other hand, in the systems usingloop primers, amplification could be observed at about between 10minutes and 20 minutes, when combined with LPF-1101 and LPR-1101 (FIG.14), LPF-1101 and LPR-1102 (FIG. 15), LPF-1101 and LPR-1104 (FIG. 17),LPF-1102 and LPR-1101 (FIG. 18) or LPF-1103 and LPR-1101 (FIG. 19). Whencombined with LPF-1101 and LPR-1103 (FIG. 16), amplification could beobserved at about 20 minutes

Test Example 2-3 Effect of Primer Sets (Group D) for Detecting HumanCK19

This test was conducted in order to examine an amplification patternwhen RNA of human CK19 was measured by the RT-LAMP method using a primerset selected from Group D shown in Example 2-2.

1) Primer Set (Group D)

Examination was carried out using a primer set consisting of FIP:FA-601, RIP: RA-604, F3: F3-601 and R3: R3-601, and a primer set incombination with various loop primers: LPF-601 and LPR-601.

2) Preparation of Human CK19RNA Sample

Following the operation as shown in Test example 2-1, samples wereprepared so that the copy numbers were 60000, 6000, 600, 60 and 0(control), respectively.

3) Composition of Reaction Solution, 4) RT-LAMP Method and 5)Observation of Amplification were Conducted in a Similar Manner as TestExample 2-1.

6) Results

Results obtained for the primer set not including loop primers and theprimer set including various loop primers are shown in FIG. 20 and FIG.21, respectively. These results show that when loop primers are notused, the time required until amplification of nucleic acid can beobserved was about 70 minutes. Additionally, even for the copy number of0, non-specific amplification was observed, suggesting slightly lowspecificity. On the other hand, when loop primers were used,amplification could be observed at about 20 minutes, and non-specificamplification was not observed for the copy number of 0. Specificity wasimproved by the use of loop primers.

Test Example 2-4 Measurement Sensitivity

This test was conducted in order to examine the sensitivity ofmeasurement for human CK19RNA when the measurement was conducted usingeach primer set selected from the Group A, C and D shown in Testexamples 2-1 to 2-3, or a primer set selected from Group B.

1) Primer Set

A group: FIP: FA-401, RIP: RA-401, F3: F3-401, R3: R3-401,

Loop primer: LPF-401, LPR-404

B group: FIP: FA1-EK, RIP: RA6-EK, F3: F3-EK, R3: R3-EK,

Loop primer: LPF1-EK, LPR2-EK

C group: FIP: FA-1101, RIP: RA-1101, F3: F3-1101, R3: R3-1101,

Loop primer: LPF-1101, LPR-1101

D group: FIP: FA-601, RIP: RA-604, F3: F3-601, R3: R3-601,

Loop primer: LPF-601, LPR-601

2) Preparation of Human CK19RNA Sample

Following the operation as shown in Test example 2-1, samples wereprepared so that the copy numbers were 60000, 20000, 6000, 2000, 600,200, 60, 20 and 0 (control), respectively.

3) Composition of Reaction Solution, 4) RT-LAMP Method and 5)Observation of Amplification were Conducted in a Similar Manner as TestExample 2-1.

6) Results

Results are shown in FIGS. 22 to 25. These results show that in everygroup of primer set, the larger the number of copies, the shorter thetimer required until amplification of nucleic acid can be observed, andamplification was observed at about 15 to 25 minutes when the copynumber was 60000. As for Group A, amplification was observed at 20 to 30minutes when the copy number was 200 or more (FIG. 22); as for Group B,amplification was observed at 15 to 20 minutes when the copy number was200 or more (FIG. 23), as for Group C, amplification was observed at 15to 25 minutes when the copy number was 600 or more (FIG. 24); and as forGroup D, amplification was observed at 25 to 35 minutes when the copynumber was 200 or more (FIG. 25).

Test Example 2-5 Amplification Specificity to Human CK19RNA

Amplification specificity to human CK19RNA was examined when measurementwas conducted using each primer set shown in Test example 2-4. It isknown that cytokeratins (CK) have isoforms such as human CK18, 20 andthe like besides human CK19. These isoforms comprises a base sequencehaving about 60% homology with human CK19. This test was conducted fordetermining whether human CK19 could be tested distinguishably fromhuman CK18 or 20 when measurement was conducted by using the aboveprimer sets.

1) Primer Set

Examination was made for the same primer sets as used in Test example2-4.

2) Preparation of RNA Sample

Human CK19RNA samples were prepared so that the copy numbers were 60000,6000, 600 and 0 (control) by conducting operation similar to that ofTest example 2-1. As to RNAs of human CK18 and human CK20, samples wereprepared in a similar manner so that the respective copy numbers were60000.

3) Composition of Reaction Solution, 4) RT-LAMP Method and 5)Observation of Amplification were Conducted in a Similar Manner as TestExample 2-1.

6) Results

FIGS. 26 to 29 show results using each primer set shown in Test example2-4. These results show that human CK19RNA, but absolutely not humanCK18RNA and human CK20RNA are amplified when any group of primer set isused.

Test Example 2-6 Observation of Amplification with Respect to HumanCK19RNA (Measurement of Turbidity)

1) Primer Set (Group C)

Examination was made for a primer set using FIP:FA-1101, RIP:RA-1101,F3:F3-1101, R3:R3-1101 and loop primers: LPF-1101 and LPR-1101 incombination.

2) Preparation of Human CK19RNA Sample

Samples were prepared so that the copy numbers were 100000, 10000, 1000and 0 (control) by conducting operation similar to that of Test example2-1.

3) Composition of Reaction Solution dNTPs (manufactured by GIBCO) 0.4 mMMgSO₄ 2 mM Dithiothreitol 5 mM Betaine) (manufactured by Sigma) 640 mMThermopol buffer (manufactured by New England BioLabs) AMV reversetranscriptase (manufactured by Promega) 1.25 U Bst DNA polymerase(manufactured by New 16 U England BioLabs) Tergitol (manufactured bySigma) 1% Primer FIP 40 pmol, RIP 40 pmol, F3 primer  5 pmol, R3 primer 5 pmol, Loop primer(loop F, loop R) each 20 pmol Tris HCl(pH 8) 60 mM4) RT-LAMP Method was Conducted in the Manner as Described in TestExample 2-1. The Reaction was Allowed for 1.5 Hour in a 0.2 mL Tube at65° C.5) Observation of Amplification

Turbidity of the RT-LAMP reaction was determined over time fromabsorbance at a wavelength of 500 nm. As a measuring machine, LA-200manufactured by Teramecs was used.

6) Results

Results are shown in FIG. 30. Depending on the copy number of template,starting of amplification was observed between 11 and 13 minutes afterstarting the measurement.

Test Example 2-7 Observation of Amplification with Respect to HumanCK19RNA (Measurement of Turbidity)

1) Primer Set (Group C)

Examination was made for a primer set using FIP:FA-1101, RIP:RA-1101,F3:F3-1101, R3:R3-1101 and loop primers: LPF-1101 and LPR-1101 incombination, and for the primer set added with RNase inhibiter.

2) Preparation of Human CK19RNA Sample

Samples were prepared so that the copy numbers were 100000, 10000, 1000and 0 (control) by conducting operation similar to that of Test example2-1.

3) Composition of Reaction Solution

A reaction solution having a similar composition as that of Test example2-6 was used except that 25 U of RNase Inhibitor (manufactured byTOYOBO) was added.

4) RT-LAMP Method and 5) Observation of Amplification were Conducted ina Similar Manner as Test Example 2-6.

6) Results

Results are shown in FIG. 31. Depending on the copy number of template,starting of amplification was observed between 10 and 12 minutes afterstarting the measurement. By adding RNase inhibitor, influence ofsubstances that inhibit amplification reaction was reduced, so that thetime required until amplification can be observed was reduce.

Test Example 3-1

This test was conducted in order to examine the time required untilamplification could be observed from he starting of the reaction foreach primer set, measured by the RT-LAMP method using the followingcombinations of primers from the various primers shown in Example 3-2.

1) Method for Preparing Human CK20 RNA Sample

By conducting RT-PCR using a primer designed based on a base sequence ofhuman CK20, human CK20cDNA was isolated from total RNA derived fromKATOIII (stomach cancer cell). From human CK20cDNA cloned intopBluescript (plasmid manufactured by STRATAGENE), a transcriptionproduct was synthesized using in vitro transcription system (Riboprobein vitro transcription system (manufactured by Promega)). RNAconcentration of the undiluted solution thus obtained was determined bymeasuring absorbance at 260 nm, and based on the result, the solutionwas diluted in 50 ng/μl yeast RNA (manufactured by Ambion) so that thecopy number of RNA of human CK20 was 600000, which were used as templatesolutions.

2) Primer Set

A variety of primers were used in the combinations as shown in Table 2.TABLE 2 Primer set and time required until amplification can be observedFIP RIP F3 primer R3 primer Time (min) KFA-5 KRA-5 KF3-5 KR3-5 25 KFA-5aKRA-5 KF3-5 KR3-5 26 KFA-5b KRA-5 KF3-5 KR3-5 23 KFA-5d KRA-5 KF3-5KR3-5 26 KFA-5e KRA-5 KF3-5 KR3-5 25 KFA-5f KRA-5 KF3-5 KR3-5 24 KFA-5KRA-5a KF3-5 KR3-5 30 KFA-5 KRA-5c KF3-5 KR3-5 35 KFA-5 KRA-5d KF3-5KR3-5 30 KFA-5 KRA-5e KF3-5 KR3-5 36 KFA-5 KRA-5f KF3-5 KR3-5 403) Composition of Reaction Solution

A similar composition with that of Test example 1-1 was used, and loopprimers were not added.

4) RT-LAMP Method

To 23 μl of reaction solution containing the above four of primers, 2 μlof RNA sample of human CK20 was added, and heated at 65° C. for onehour.

5) Observation of Amplification

Measurement was conducted in real time in a similar manner with Testexample 1-1.

6) Results

The time required until amplification could be observed when reactionwas conducted using each primer set is shown in Table 2. These resultsshow that the maximum time is 40 minutes and amplification was confirmedin 30 minutes in most of the primer sets.

Test Example 3-2

This test was conducted in order to examine the sensitivity whenmeasurement by RT-LAMP method was conducted using a primer set requiringa short time for observation of amplification selected from the primersets examined in Test example 3-1, and using a primer set combined withloop primers.

1) Method for Preparing Human CK20RNA Sample

Samples were prepared so that the copy numbers were 0, 190,960,4800,24000, 120000, 600000 respectively by conducting a similaroperation as Test example 3-1.

2) Primer Set

Examination was made for the primer sets of following combinations.

Set 1: FIP (KFA-5b), RIP (KRA-5)

F3 primer (KF3-5), R3 primer (KR3-5)

Loop primers (K-LPF2, K-LPR1)

Set 2: FIP (KFA-5b), RIP (KRA-5)

F3 primer (KF3-5), R3 primer (KR3-5)

Loop primers (K-LPF2, K-LPR2)

3) Composition of Reaction Solution

A similar composition with that of Test example 1-1 was used.

4) RT-LAMP Method and 5) Observation of Amplification were Conducted ina Similar Manner as Test Example 3-1.

6) Results

The examination result as to Primer set 1 is shown in FIG. 32. Theresult shows that the larger the amount of template of human CK20RNA,the shorter the time required until amplification can be observed.However, even in the case where the amount of template was 190 copies,DNA amplification was observed in 30 minutes, and even in the case wherethe amount of template was 600000 copies, DNA amplification was observedat about 10 minutes. A similar result was obtained for the case ofPrimer set 2.

Test Example 3-3 Amplification Specificity to Human CK20RNA

Amplification specificity to human CK20 when measurement was conductedusing the primer sets selected in Test example 3-2 was examined. It isknown that cytokeratins (CK) have isoforms such as human CK18, 19 andthe like besides human CK20. These isoforms comprises a base sequencehaving about 60% homology with human CK20. This test was conducted fordetermining whether human CK20 could be tested distinguishably fromhuman CK18 or 19 when measurement was conducted by using primer setsaccording to the present invention.

1) Method for Preparing RNA Sample

An RNA sample of human CK20 having a copy number of 600000 was preparedin a similar manner as described in Test example 3-1. Also as to RNAs ofhuman CK18 and human CK19, samples were prepared in a similar manner.

2) Primer Set

Primer sets 1 and 2 selected in Test example 3-2 were used.

3) Composition of Reaction Solution, 4) RT-LAMP Method, and 5)Observation of Amplification were Conducted in a Similar Manner as TestExample 3-2.

6) Results

An examination result as to Primer set 1 is shown in FIG. 33. The resultshows that human CK20RNA, but absolutely not human CK18RNA and humanCK19RNA are amplified when Primer set 1 is used, revealing specificityto human CK20RNA. A similar result was obtained for the case of Primerset 2.

Test Example 3-4 Measurement Sensitivity

This test was conducted in order to examine the sensitivity ofmeasurement for human CK20RNA when the measurement was conducted usingeach primer set.

1) Method for Preparing Human CK20RNA Sample

Samples were prepared so that the copy numbers were 0, 190, 960, 4800,24000, 120000, 600000 respectively by conducting a similar operation asTest example 3-1.

2) Primer Set

Examination was made for the primer set of following combination.

Set 3: FIP (AFA), RIP (ARAf), F3 primer (AF3), R3 primer (AR3) Loopprimers (LPF2, LPR6)

3) Composition of Reaction Solution, 4) RT-LAMP Method and 5)Observation of Amplification were Conducted in a Similar Manner as TestExample 3-2.

6) Results

The examination results are shown in FIG. 34. The results show that thelarger the amount of template of human CK20RNA, the shorter the timerequired until amplification can be observed. However, even in the casewhere the amount of template was 190 copies, DNA amplification wasobserved in 30 minutes, and even in the case where the amount oftemplate was 600000 copies, DNA amplification was observed at about 10minutes.

Test Example 3-5

This test was conducted in order to examine amplification specificity tohuman CK20RNA when measurement was conducted using the primer setselected in Test example 3-4.

1) Method for Preparing RNA Sample

An RNA sample of human CK20 was prepared so that the copy number was600000. RNA samples of human CK18 and human CK19 were also prepared in asimilar manner.

2) Primer Set

The primer set (Set 3) selected in Test example 3-4 was selected.

3) Composition of Reaction Solution, 4) RT-LAMP Method and 5)Observation of Amplification were Conducted in a Similar Manner as TestExample 3-2.

6) Results

The results are shown in FIG. 35. These results show that human CK20RNA,but absolutely not human CK18RNA and human CK19RNA are amplified by theselected primer set.

INDUSTRIAL APPLICABILITY

As described above, it was found that when the LAMP method is conductedusing a primer or a primer set of the present invention, human CK18RNA,CK19RNA and CK20RNA can be amplified with efficiency and specificity.According to this finding, primers of the present invention enabledetection of human CK18RNA, CK19RNA and CK20RNA in short time, andreduction of time required for diagnosis of cancer metastasis to lymphnodes using nucleic acid amplifying means.

1. A primer for nucleic acid amplification for detecting cytokeratinbased on the LAMP method.
 2. The primer for nucleic acid amplificationaccording to claim
 1. wherein the cytokeratin is selected from the groupconsisting of cytokeratin 18, cytokeratin 19 and cytokeratin
 20. 3. Aprimer for nucleic acid amplification for detecting human cytokeratin 18comprising an oligonucleotide having a sequence selected from thefollowing groups 1) to 5); 1) an oligonucleotide selected from a regionof base position 270-1375 in a base sequence of SEQ ID NO: 1 and acomplementary strand region thereof, containing at least 5 or moresuccessive bases in SEQ ID NO.1 and/or in the complementary strandthereof, 2) an oligonucleotide having a base sequence represented by anyone of SEQ ID NOs: 2 to 341, 3) a complementary strand of theoligonucleotide according to the above 1) or 2), 4) an oligonucleotidecapable of hybridizing with the oligonucleotide according to any one ofthe above 1) to 3) under stringent conditions, and 5) an oligonucleotidecomprising a base sequence obtainable from the oligonucleotidesaccording to the above 1) to 4) wherein one to several bases arereplaced, inserted, added or otherwise mutated, the oligonucleotidehaving a primer function.
 4. A primer for nucleic acid amplification fordetecting human cytokeratin 18, comprising an oligonucleotide selectedfrom the base sequences of SEQ ID NOs: 66 to 88 or 179 to
 341. 5. Theprimer for nucleic acid amplification according to claim 3, whereinnucleic acid amplification is conducted by the LAMP method.
 6. A primerset for nucleic acid amplification for detecting human cytokeratin 18,wherein at least two kinds of primers are selected from primers fornucleic acid amplification, each primer comprising an oligonucleotidecomprising a sequence selected from the following groups 1) to 4); 1) anoligonucleotide selected from a region of base position 270-1375 in abase sequence of SEQ ID NO: 1 and a complementary strand region thereof,containing at least 5 or more successive bases in SEQ ID NO.1 and/or inthe complementary strand thereof, 2) an oligonucleotide having a basesequence represented by any one of SEQ ID NOs: 2 to 341, 3) acomplementary strand of the oligonucleotide according to the above 1) or2), 4) an oligonucleotide capable of hybridizing with theoligonucleotide according to any one of the above 1) to 3) understringent conditions, and 5) an oligonucleotide comprising a basesequence obtainable from the oligonucleotides according to the above 1)to 4) wherein one to several bases are replaced, inserted, added orotherwise mutated, the oligonucleotide having a primer function.
 7. Theprimer set for nucleic acid amplification for detecting humancytokeratin 18 according to claim 6, wherein the nucleic acidamplification is conducted by the LAMP method.
 8. The primer set fornucleic acid amplification for detecting human cytokeratin 18 accordingto claim 7, wherein at least four kinds of primers are selected from theprimers for nucleic acid amplification each comprising anoligonucleotide.
 9. The primer set for nucleic acid amplification fordetecting human cytokeratin 18 according to claim 6, wherein at leasttwo kinds of primers included in the primer set respectively recognizetwo regions of the base sequence of SEQ ID NO: 1 and/or a complementarystrand thereof.
 10. The primer set according to claim 7, wherein theprimers included in said primer set recognize at least 6 regions in thebase sequence of SEQ ID NO: 1 and/or the complementary strand thereof.11. A primer set comprising a combination of primers, one of the primersbeing selected from (a) SEQ ID NOs: 234 to 286, the other selected from(b) SEQ ID NOs: 287 to 341, among the primers for nucleic acidamplification for detecting human cytokeratin 18, each comprising anoligonucleotide selected from the base sequences of SEQ ID NOs:234 to341.
 12. The primer set according to claim 11, further comprising acombination of primers, one of the primers being selected from (c) SEQID NOs: 66 to 88, the other selected from (d) SEQ ID NOs: 179 to 201,among the primers for nucleic acid amplification for detecting humancytokeratin 18 each comprising an oligonucleotide selected from the basesequences of SEQ ID NOs: 66 to 88 or SEQ ID NOs: 179 to
 201. 13. Theprimer set according to claim 11, further comprising a combination ofprimers, one of the primers being selected from (e) SEQ ID NOs: 202 to219, the other selected from (f) SEQ ID NOs: 220 to 233, among theprimers for nucleic acid amplification for detecting human cytokeratin18 each comprising an oligonucleotide selected from the base sequencesof SEQ ID NOs: 202 to
 233. 14. The primer set for nucleic acidamplification for detecting cytokeratin 18 comprising either one of thefollowing groups 1) to 4); 1) a primer set including oligonucleotideshaving base sequences of SEQ ID NOs: 234, 287, 66 and 179 as primers, 2)a primer set including oligonucleotides having base sequences of SEQ IDNOs: 252, 297, 68 and 182 as primers, 3) a primer set includingoligonucleotides having base sequences of SEQ ID NOs: 259, 307, 72 and184 as primers, 4) a primer set including oligonucleotides having basesequences of SEQ ID NOs: 278, 331, 79 and 193 as primers.
 15. The primerset for nucleic acid amplification for detecting human cytokeratin 18comprising either one of the following groups 1) to 4); 1) a primer setincluding oligonucleotides having base sequences of SEQ ID NOs: 234,287, 66, 179, 203 and 220 as primers, 2) a primer set includingoligonucleotides having base sequences of SEQ ID NOs: 252, 297, 68, 182,211 and 223 as primers, 3) a primer set including oligonucleotideshaving base sequences of SEQ ID NOs: 259, 307, 72, 184, 212 and 226 asprimers, 4) a primer set including oligonucleotides having basesequences of SEQ ID NOs: 278, 331, 79, 193, 214 and 228 as primers. 16.The primer set for nucleic acid amplification for detecting humancytokeratin 18 comprising either one of the following groups 1) to8); 1) a primer set including oligonucleotides having base sequences ofSEQ ID NOs: 280, 334, 82 and 195 as primers, 2) a primer set includingoligonucleotides having base sequences of SEQ ID NOs: 281, 335, 83 and196 as primers, 3) a primer set including oligonucleotides having basesequences of SEQ ID NOs: 282, 336, 84 and 197 as primers, 4) a primerset including oligonucleotides having base sequences of SEQ ID NOs: 282,337, 84 and 197 as primers, 5) a primer set including oligonucleotideshaving base sequences of SEQ ID NOs: 283, 338, 85 and 198 as primers, 6)a primer set including oligonucleotides having base sequences of SEQ IDNOs: 284, 339, 86 and 199 as primers, 7) a primer set includingoligonucleotides having base sequences of SEQ ID NOs: 285, 340, 87 and200 as primers, 8) a primer set including oligonucleotides having basesequences of SEQ ID NOs: 286, 341, 88 and 201 as primers.
 17. The primerset for nucleic acid amplification for detecting human cytokeratin 18comprising either one of the following groups 1) to 6); 1) a primer setincluding oligonucleotides having base sequences of SEQ ID NOs: 282,336, 84, 197, 216 and 229 as primers, 2) a primer set includingoligonucleotides having base sequences of SEQ ID NOs: 282, 337, 84, 197,216 and 230 as primers, 3) a primer set including oligonucleotideshaving base sequences of SEQ ID NOs: 282, 337, 84, 197, 216 and 231 asprimers, 4) a primer set including oligonucleotides having basesequences of SEQ ID NOs: 283, 338, 85, 198, 217 and 232 as primers, 5) aprimer set including oligonucleotides having base sequences of SEQ IDNOs: 286, 341, 88, 201, 218 and 233 as primers, 6) a primer setincluding oligonucleotides having base sequences of SEQ ID NOs: 286,341, 88, 201, 219 and 233 as primers.
 18. A method for detecting nucleicacids of human cytokeratin 18 using a necessary primer selected from theprimers according to claim 3, or a primer set selected from the primersets.
 19. A primer for nucleic acid amplification for detecting humancytokeratin 19 comprising an oligonucleotide having a sequence selectedfrom the following groups 1) to 5); 1) an oligonucleotide selected froma region of base position 270-930 in a base sequence of SEQ ID NO: 342and a complementary strand region thereof, containing at least 5 or moresuccessive bases in SEQ ID NO.1 and/or in the complementary strandthereof, 2) an oligonucleotide having a base sequence represented by anyone of SEQ ID NOs: 343 to 432, 3) a complementary strand of theoligonucleotide according to the above 1) or 2), 4) an oligonucleotidecapable of hybridizing with the oligonucleotide according to any one ofthe above 1) to 3) under stringent conditions, and 5) an oligonucleotidecomprising a base sequence obtainable from the oligonucleotidesaccording to the above 1) to 4) wherein one to several bases arereplaced, inserted, added or otherwise mutated, the oligonucleotidehaving a primer function.
 20. A primer for nucleic acid amplificationfor detecting human cytokeratin 19, comprising an oligonucleotideselected from the base sequences of SEQ ID NOs: 357 to 361 or 378 to434.
 21. The primer for nucleic acid amplification for detecting humancytokeratin 19 according to the claim 19, wherein the nucleic acidamplification is conducted by the LAMP method;
 22. A primer set fornucleic acid amplification for detecting human cytokeratin 19, whereinat least two kinds of primers are selected from primers for nucleic acidamplification, each primer comprising an oligonucleotide comprising asequence selected from the following groups 1) to 5); 1) anoligonucleotide selected from a region of base position 270-930 in abase sequence of SEQ ID NO: 342 and a complementary strand regionthereof, containing at least 5 or more successive bases in SEQ ID NO.342and/or in the complementary strand thereof, 2) an oligonucleotide havinga base sequence represented by SEQ ID NOs: 343 to 434, 3) acomplementary strand of the oligonucleotide according to the above 1) or2), 4) an oligonucleotide capable of hybridizing with theoligonucleotide according to any one of the above 1) to 3) understringent conditions, and 5) an oligonucleotide comprising a basesequence obtainable from the oligonucleotides according to the above 1)to 4) wherein one to several bases are replaced, inserted, added orotherwise mutated, the oligonucleotide having a primer function.
 23. Theprimer set for detecting human cytokeratin 19 according to claim 22,wherein the nucleic acid amplification is conducted by the LAMP method.24. The primer set for nucleic acid amplification for detecting humancytokeratin 19 according to claim 22, wherein at least four kinds ofprimers are selected from the primers for nucleic acid amplificationeach comprising an oligonucleotide.
 25. The primer set for nucleic acidamplification for detecting human cytokeratin 19 according to any one ofclaims 22, wherein at least two kinds of primers included in the primerset respectively recognize two regions of the base sequence of SEQ IDNO: 342 and/or a complementary strand thereof.
 26. The primer set fordetecting human cytokeratin 19 according to claim 22, wherein theprimers included in said primer set recognize at least six regions inthe base sequence represented by SEQ ID NO: 342 and/or a complementarystrand thereof.
 27. A primer set comprising a combination of primers,one of the primers being selected from (a) SEQ ID NOs: 413 to 417, 419,422, or 424 to 427, the other selected from (b) SEQ ID NOs: 418, 420,421, 423 or 428 to 434, among the primers for nucleic acid amplificationfor detecting human cytokeratin 19, each comprising an oligonucleotideselected from the base sequences of SEQ ID NOs: 413 to
 434. 28. Theprimer set according to claim 27, further comprising a combination ofprimers, one of the primers being selected from (c) SEQ ID NOs: 357 to361, the other selected from (d) SEQ ID NOs: 378 to 384, among theprimers for nucleic acid amplification for detecting human cytokeratin19 each comprising an oligonucleotide selected from the base sequencesof SEQ ID NOs: 357 to 361 or SEQ ID NOs: 378 to
 384. 29. The primer setaccording to claim 27, further comprising a combination of primers, oneof the primers being selected from (e) SEQ ID NOs: 385 to 398, the otherselected from (f) SEQ ID NOs: 399 to 412, among the primers for nucleicacid amplification for detecting human cytokeratin 19 each comprising anoligonucleotide selected from the base sequences of SEQ ID NOs: 385 to412.
 30. A primer set comprising either one of the followingcombinations 1) to 4); 1) combination of primers selected respectivelyone from (a) SEQ ID NO: 413 to 417, (b) SEQ ID NO: 418, (c) SEQ ID NO:357 and (d) SEQ ID NO: 378, 2) combination of primers selectedrespectively one from (a) SEQ ID NO: 419, (b) SEQ ID NO: 420 to 421, (c)SEQ ID NO: 358 and (d) SEQ ID NO: 379, 3) combination of primersselected respectively one from (a) SEQ ID NO: 422, (b) SEQ ID NO: 423,(c) SEQ ID NO: 359 and (d) SEQ ID NO: 380, 4) combination of primersselected respectively one from (a) SEQ ID NO: 424 to 427, (b) SEQ ID NO:428 to 434, (c) SEQ ID NO: 360 to 361 and (d) SEQ ID NO: 381 to 384,among primers for nucleic acid amplification for detecting humancytokeratin 19 each comprising an oligonucleotide having a base sequencerepresented by each SEQ ID NO:.
 31. A primer set comprising either oneof the following combinations 1) to 4); 1) combination of primersselected respectively one from (a) SEQ ID NO: 413 to 417, (b) SEQ ID NO:418, (c) SEQ ID NO: 357, (d) SEQ ID NO: 378, (e) SEQ ID NO: 385 to 391and (f) SEQ ID NO: 399 to 402, 2) combination of primers selectedrespectively one from (a) SEQ ID NO: 419, (b) SEQ ID NO: 420 to 421, (c)SEQ ID NO: 358, (d) SEQ ID NO: 379, (e) SEQ ID NO: 392 to 393 and (f)SEQ ID NO: 403 to 406, 3) combination of primers selected respectivelyone from (a) SEQ ID NO: 422, (b). SEQ ID NO: 423, (c) SEQ ID NO: 359,(d) SEQ ID NO: 380, (e) SEQ ID NO: 394 to 396 and (f) SEQ ID NO: 407 to409, 4) combination of primers selected respectively one from (a) SEQ IDNO: 424 to 427, (b) SEQ ID NO: 428 to 434, (c) SEQ ID NO: 360 to 361,(d) SEQ ID NO: 381 to 384, (e) SEQ ID NO: 397 to 398 and (f) SEQ ID NO:411 to
 412. 32. A primer set for nucleic acid amplification fordetecting human cytokeratin 19 comprising either one of the followinggroups 1) to 3); 1) a primer set including oligonucleotides having basesequences represented by SEQ ID NOs: 413, 418, 357 and 378 as primers,2) a primer set including oligonucleotides having base sequencesrepresented by SEQ ID NOs: 419, 421, 358 and 379 as primers, 3) a primerset including oligonucleotides having base sequences represented by SEQID NOs: 424, 431, 360 and 381 as primers.
 33. A primer set for nucleicacid amplification for detecting human cytokeratin 19 comprising eitherone of the following groups 1) to 4); 1) a primer set includingoligonucleotides having base sequences represented by SEQ ID NOs: 413,418, 357, 378, 385 and 402 as primers, 2) a primer set includingoligonucleotides having base sequences represented by SEQ ID NOs: 419,421, 358, 379, 392 and 404 as primers, 3) a primer set includingoligonucleotides having base sequences represented by SEQ ID NOs: 422,423, 359, 380, 394 and 407 as primers, 4) a primer set includingoligonucleotides having base sequences represented by SEQ ID NOs: 424,431, 360, 381, 397 and 411 as primers.
 34. A method for detectingnucleic acids using a necessary primer selected from the primersaccording to claim 19, or a primer set selected from the primer sets.35. A primer for nucleic acid amplification for detecting humancytokeratin 20 comprising an oligonucleotide having a sequence selectedfrom the following groups 1) to 5); 1) an oligonucleotide selected froma region of base position 340-490 or a region of base position 495-1050in a base sequence of SEQ ID NO: 435 and regions of a complementarystrand thereof, containing at least 5 or more successive bases in SEQ IDNO.435 and/or in the complementary strand thereof, 2) an oligonucleotidehaving a base sequence represented by any one of SEQ ID NOs: 436 to 460,3) a complementary strand of the oligonucleotide according to theabove 1) or 2), 4) an oligonucleotide capable of hybridizing with theoligonucleotide according to any one of the above 1) to 3) understringent conditions, and 5) an oligonucleotide comprising a basesequence obtainable from the oligonucleotides according to the above 1)to 4) wherein one to several bases are replaced, inserted, added orotherwise mutated, the oligonucleotide having a primer function.
 36. Aprimer for nucleic acid amplification for detecting human cytokeratin20, comprising an oligonucleotide selected from the base sequences ofSEQ ID NOs: 443, 444 or 454 to
 474. 37. The primer for nucleic acidamplification for detecting human cytokeratin 20 according to claim 35,wherein the nucleic acid amplification is conducted by the LAMP method.38. A primer set for nucleic acid amplification for detecting humancytokeratin 20, wherein at least two kinds of primers are selected fromprimers for nucleic acid amplification, each primer comprising anoligonucleotide comprising a sequence selected from the followinggroups 1) to 5); 1) an oligonucleotide selected from a region of baseposition 340-1050 in a base sequence of SEQ ID NO: 435 and acomplementary strand region thereof, containing at least 5 or moresuccessive bases in SEQ ID NO.435 and/or in the complementary strandthereof, 2) an oligonucleotide having a base sequence represented by anyone of SEQ ID NOs: 436 to 460, 3) a complementary strand of theoligonucleotide according to the above 1) or 2), 4) an oligonucleotidecapable of hybridizing with the oligonucleotide according to any one ofthe above 1) to 3) under a stringent condition, and 5) anoligonucleotide comprising a base sequence obtainable from theoligonucleotides according to the above 1) to 4) wherein one to severalbases are replaced, inserted, added or otherwise mutated, theoligonucleotide having a primer function.
 39. The primer set fordetecting human cytokeratin 20 according to claim 38, wherein thenucleic acid amplification is conducted by the LAMP method.
 40. Theprimer set for nucleic acid amplification for detecting humancytokeratin 20 according to claim 39, wherein at least four kinds ofprimers are selected from the primers for nucleic acid amplificationeach comprising an oligonucleotide.
 41. The primer set for nucleic acidamplification for detecting human cytokeratin 20 according to claim 38,wherein at least two kinds of primers included in the primer setrespectively recognize two regions of the base sequence of SEQ ID NO:435 and/or a complementary strand thereof.
 42. The primer set fordetecting human cytokeratin 20 according to claim 38, wherein theprimers included in said primer set recognize at least six regions inthe base sequence represented by SEQ ID NO:435 and/or a complementarystrand thereof.
 43. A primer set comprising a combination of primers,one of the primers being selected from (a) SEQ ID NOs: 461 to 466, theother selected from (b) SEQ ID NOs: 468 to 473, among the primers fornucleic acid amplification for detecting human cytokeratin 20 eachcomprising an oligonucleotide selected from the base sequences of SEQ IDNOs: 461 to 466 or SEQ ID NOs: 468 to
 473. 44. The primer set fordetecting human cytokeratin 20 according to claim 43, further comprisingoligonucleotides having base sequences represented by SEQ ID NO: 444 andSEQ ID NO: 455 as primers.
 45. The primer set for detecting humancytokeratin 20 according to claim 43, further comprising anoligonucleotide having a base sequence represented by SEQ ID NO: 457and/or 459 or 460 as a primer.
 46. A primer set for detecting humancytokeratin 20 comprising oligonucleotides having base sequencesrepresented by SEQ ID NOs: 443, 454, 467 and 474 as primers.
 47. Aprimer set for detecting human cytokeratin 20, wherein anoligonucleotide having abase sequence represented by SEQ ID NO: 456and/or 458 is used as a primer in addition to oligonucleotides havingbase sequences represented by SEQ ID NOs: 443, 454, 467 and
 474. 48. Amethod for detecting nucleic acids using a necessary primer selectedfrom the primers according to claim 35, or a primer set selected fromthe primer sets.
 49. The method for detecting nucleic acids according tothe above 18, wherein the nucleic acid amplification is conducted by theLAMP method.
 50. A reagent for use in the method for detecting nucleicacids according to claim
 18. 51. A reagent kit for use in the method fordetecting nucleic acids according to claim
 18. 52. A nucleic aciddetecting system using the method for detecting nucleic acids accordingto claim 18.